Capstone Project

Name of Student:_Neeraj Kumar Yadav

Registration Number:10807700 Section No: B18E4 Roll No.63

Internal Faculty Advisor :Dr.Kuldip C Verma

Title of the Project: Biochemical Analysis of Active intergradient’s in

Asparagus Racemosus (Shatavari) and its various uses

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Contents

1-Objective

2-Abstract

3-Introduction

4- Asparagus Racemosus : the plant species

5-Geographical distribution :

6-Active constituents of Asparagus Racemosus

7-Shatavarin production from in vitro cultures of Asparagus Racemosus

7A-Introduction of Shatavarin

7B- MATERIALS AND METHODS

7B-b-Plant material

7B-c- Callus induction and maintenance

7B-d-Growth studies

7B-e- Extraction of saponin

7C-EXPECTED RESULTS

7D-Studies on induction time

7E- Analysis of shatavarin

7F-DISCUSSION

8-Asparagus Racemosus propagation methods through tissue culture technique

8A-Explant Source

8B-Plant cell culture

8C-Maintenance of Aseptic Environment

8D-Murashige-Skoog Medium Composition (Table 1)

8E-Culture Medium :

8F-Surface sterilization of explants

8G-Inoculation and Incubation

8H-Regeneration was carried out in following stages

8H-a-Induction of multiple shoot buds and callus

8H-b-Proliferation of multiple shoot buds and callus

8I-Hardening of plantlets and pot transfer

9- Pharmacological applications of Asparagus Racemosus

9A-Effect on neurodegenerative disorders

9B-Anti-diarrhoeal effects

9D-Anti-dyspepsia effects

9E-Cardio protective effects

9F-Anti-bacterial effects

9G-Immunoadjuvant effects

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9H-Antitussive effects

10-Challenges in conservation and sustainable use of Asparagus Racemosus

11-Cultivation practices

11A-Organic cultivation

11B-Post-harvest handling

12 -Uses & Benefits of Asparagus

13-Qualitative phytochemical analysis

13a-Tests for carbohydrates

13b-Tests for alkaloids

13c-Tests for proteins and amino acids

13d-Tests for tannins and phenolics

13-eTest for flavonoids

13f-Test for triterpenoids

13g-Tests for steroids

13h-Test for saponins

13i-Tests for fixed oils

13j-Tests for glycosides

13k-Test for gums and mucilages

14-Literature Review

15-Conclusion

16-Reference

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1-Objective

1-Biochemical analysis of different parts of the plant.

2-Plant Tissue Culture Technique of Asparagus Racemosus.

(http://classicaurvedic.blogspot.com/2011_01_01_archive.html)

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2-Abstract

Asparagus Racemosus is an important medicinal plant of tropical and subtropical India. Its medicinal usage has been reported in the Indian and British Pharmacopoeias and in traditional systems of medicine such as Ayurveda, Unani and Siddha. Asparagus racemosus is one of the important medicinal plants found in India, China and other parts of the world. This plant is known to produce steroidal saponins called Shatavarins. Callus cultures of A. racemosus were initiated in a modified MS medium supplemented with 1.0 mg/L NAA and 2, 4-D and 0.5 mg/L BAP and compared for growth and production of saponin over a period of 60 days. Saponin production was evaluated at a regular interval of 15 days. Root calli produces more saponin compared to nodal calli and maximum accumulation was found to be 10.38 ± 0.14 mg/g of callus after 60 days of inoculation. Total saponins from the nodal calli were found to be 7.69 ± 0.136 mg/g of callus. Compared to wild type roots, in vitro cultures showed 20 fold increases in shatavarin levels. High performance liquid chromatography (HPLC) chromatograms of the cultures indicated that the overall saponin profile of in vitro and in vivo plant root extract is similar.

3-Introduction

The World Health Organization (2003) has estimated that 80% of the population of developing countries being unable to afford pharmaceutical drugs rely on traditional medicines, mainly plant based, to sustain their primary health care needs. India is one of the most medico-culturally diverse countries in the world where the medicinal plant sector is part of a time-honoured tradition that is respected even today. Here, the main traditional systems of medicine include Ayurveda, Unani and Siddha. The earliest mention of the use of plants in medicine is found in the Rigveda which was written between 4500 and 1600 BC. It is however in Ayurveda that the specific properties of plants and their use as medicinal drugs has been dealt with in great detail. ‘Ayurveda’ literally translated means science of life . Ananthacharya (1939) in defining this system of medicine said Ayurveda scrutinizes the subtle process of life, studies its nature, ways and conditions of development and deduces there-from a universal course of conduct for man’s guidance in life . Ayurveda has eight divisions dealing with different aspects of the art of healing. These include kaya cikitsa (internal medicine), salya tantra (surgery), salakya tantra (treatment of diseases of the head and neck region), agada tantra (toxicology), bhuta vidya (management of mental ailments), bala tantra (pedi-atrics), rasayana tantra (rejuvenation therapy and geriatrics) and vajikarana tantra (science of aphrodisiacs). Around 1250 plants are presently used in various Ayurvedic formulations. Asparagus Racemosus Willd. is one such important medicinal plant which is regarded as a ‘rasayana’ (plant drugs promoting general well being by

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increasing cellular vitality and resistance) in the Ayurvedic system of medicine .Asparagus Racemosus is an important medicinal plant of tropical and subtropical India. Its medicinal usage has been reported in the Indian and British Pharmacopoeias and in indige-nous systems of medicine. The genus Asparagus includes about 300 species around the world. The genus is considered to be medicinally important because of the presence of steroidal saponins and sapogenins in various parts of the plant. Out of the 22 species of Asparagus recorded in India; Aspara-gus racemosus is the one most commonly used in traditional medicine.

4- Asparagus Racemosus : the plant species

Asparagus Racemosus Willd. (family Asparagaceae; Lili-aceae), is commonly called Satavari, Satawar or Satmuli in Hindi; Satavari in Sanskrit; Shatamuli in Bengali; Shatavari or Shatmuli in Marathi; Satawari in Gujarati; Toala-gaddalu or Pilli-gaddalu in Telegu; Shimaishadavari or Inli-chedi in Tamil; Chatavali in Malayalam; Majjigegadde or Aheruballi in Kan-nada; Kairuwa in Kumaon; Narbodh or Satmooli in Madhya Pradesh; and Norkanto or Satawar in Rajasthan .Plant grows throughout the tropical and subtropical parts of India up to an altitude of 1500 m. The plant is a spinous under-shrub, with tuberous, short rootstock bearing numerous succulent tuberous roots (30–100 cm long and 1–2 cm thick) that are silvery white or ash coloured externally and white inter-nally. These roots are the part that finds use in various medicinal preparations. The stem is woody, climbing, whitish grey or brown coloured with small spines. The plant flowers during February–March leaving a mild fragrance in its surrounding and by the end of April, fruits can be seen with attractive red berries.

5-Geographical distribution :Asparagus Racemosus is distributed throughout tropical Africa, Java, Australia, India, Srilanka and Southern parts of China. In India, It is found in tropical, sub tropical regions and in Himalayas upto 1000 to 1500 m.

6-Active constituents of Asparagus Racemosus

The major active constituents of Asparagus Racemosus are steroidal saponins (Shatavarins I–IV) that are present in the roots. Shatavarin IV is a glycoside of sarsasapogenin having two molecules of rhamnose and one molecule of glucose . Other active compounds such as quercetin, rutin (2.5% dry basis) and hyperoside are found in the flowers and fruits; while diosgenin and quercetin-3 glucuronide are present in the leaves. Synthesis of sarsasapogenin in the callus cultures of Asparagus Racemosus was also reported earlier by Kar and Sen (1985). DPPH (α ,α-diphenyl-β-picrylhydrazyl) autography-directed separation resulted in the identification of a new antioxidant compound from Asparagus Racemosus named ‘racemofuran’. Previously, the isolation and spectral data of a new isoflavone, 8-methoxy-5,6,4-trihydroxyisoflavone 7-o -β -d -glucopyranoside, was reported from the roots of the plant. Later, a new 9, 1-dihydrophenenthrene derivative named ‘Racemosol’ was isolated from the ethanol extract of

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roots (Sekine et al., 1997). Its structure was elucidated by spectroscopic analysis as 9, 10-dihydro-1, 5-dimethoxy-8-methyl-2, 7-phenenthrenediol. Also, sarsasapogenin and kaempferol have been isolated from the woody portion of tuberous roots of Asparagus Racemosus . These compounds were identified on the basis of chemical and spectroscopic evidence .

7-Shatavarin production from in vitro cultures of Asparagus Racemosus

7A-Introduction of Shatavarin

Asparagus Racemosus (local name Shatavari) is an important monocot medicinal plant. It is routinely used in indigenous medicines and is one of the extensively exported medicinal formulations from India for menopausal and fertility related problems. Steroidal saponins Shatavarins , the major bioactive component in A. racemosus, is particularly drawing attention because of its role as an immunomodulant, galactogauge, adaptogen, antitusive, anticarcinogen, antioxidant, antidierrhial and as a general tonic for both the sexes . Although, the entire plant is reported to contain Shatavarins, the fasciculate roots of A. racemosus are considered to be the richest source of Shatavarins. The major constraint in conventional multiplication method of A. racemosus through seed propagation is requirement of elaborate pretreatments to break the dormancy and high mortality of seedlings. The grown-up plants can be harvested only after flowering which takes 3 to 4 years because Shatavarins are accumulated in the roots only post flowering . The normal practice of root collection involves uprooting of the whole plant and random pretreatment methods, which usually leads to wastage of 50% of the collected material. Moreover, the growing demand for the plant has caused a serious reduction in native populations due to over-harvest and deforestation. Therefore, this is one of those several medicinal plants for which sustainable conservation methods are required on a priority basis. This plant has also been recognized as ‘vulnerable’ . Plant tissue culture has been successfully employed for several plants facing similar problems and can be tried out for A. racemosus also. Very few reports are available regarding callus induction in A. racemosus and its in vitro . To the best of our knowledge, no study has been undertaken to correlate the combinations of growth regulators and their effect on callus induction, morphological aspects, the biosynthetic dynamics of the cultures with respect to secondary metabolite accumulation and its effect on overall saponin profile in cell cultures. Tissue culture methods are being successfully tried out even for several monocot plants facing similar problems. In view of the foregoing, this study was undertaken to optimize the conditions for the in vitro saponin production from A. racemosus in vitro cultures.

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7B- MATERIALS AND METHODS

7B-b-Plant material

Different parts like nodal segments and roots of the field grown A. racemosus plant were collected after confirming the authenticity of the plant by an expert taxonomist from the Department of Botany, Hislop College, Nagpur, India. The plant materials were washed with detergent solution and distilled water for 30 min. These plant parts were surface sterilized inside a laminar hood firstly with 0.1% HgCl2 for 3 min and then with 70% ethanol for 5 min followed by washings with sterile distilled water. These plant parts were then dried and cut into small pieces to be used as explants for the inoculations.

7B-c- Callus induction and maintenance

An initiation medium was designed using modified MS salts supplemented with vitamins in full concentration and 3% sucrose (w/v) (Murashieg and Skoog, 1962). Three different hormones namely naphthalene acetic acid (NAA, 0 – 3.0 mg/L), 2.4- Dichlorophenoxyacetic acid (2, 4-D, 0 - 4.0 mg/L) and 6- Benzyl aminopurine (BAP, 0 - 2.0 mg/L) were used to study callus induction from nodal and root explants in various combinations. The pH of the medium was adjusted to 5.7 and 0.6% phytagel was used as the gelling agent. Medium was dispensed into tissue culture test tubes for slant preparation and autoclaved at 121°C and 15 Lb pressure for 20 min. After the inoculations of the explants, tubes were exposed to a photoperiod of 16 h of light and 8 h of darkness at 25 ± 2°C and sub cultured at regular intervals of three weeks incubation in the culture room.

7B-d-Growth studies

The fresh weight of the cultured tissues was measured by carefully removing the adhered agar cells were dried in a hot air oven to a constant weight and their dry weights were recorded. In all the experiments data is expressed as mean performance of all the replicas and their standard deviations.

7B-e- Extraction of saponin

Briefly, cells were extracted separately with methanol (1:2) overnight and procedure was repeated 4 times. All the extracts were pooled and concentrated at 60°C on a rotary evaporator to dryness. The residue was redissolved in 10 ml of H2O and further extracted with n- butanol. The n- butanol fraction was finally concentrated to dryness on a rotary

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evaporator under reduced pressure and redissolved in 5 ml of methanol and stored and analyzed using standard Shatavarin IV as the marker. These extracts were also compared with standard Sarsapogenin , which is a precursor of Shatavarins. For the preparation of standard solutions, Shatavarin IV was dissolved in methanol and was diluted to get a final concentration range of 100 to 500 gml-1. The solutions were filtered through a 0.2 µm acrodisc. Evaluation of each point was repeated 3 times at 220 nm and the calibration curve was fitted by linear regression. This calibration curve was utilized for the estimation of total Shatavarin present in the methanolic extracts from the wild type plant and in vitro extracts. RP- HPLC analysis of the Saponin samples were carried out using a Knauer smart line manager- 5000 system fitted with a C18 column (4 um, 150 mm x 3.9 mm I.D.), UV detector and 20L injection loop. Acetonitrile and 30% aqueous methanol were used as the mobile phase with gradients from 8 to 100% of Acetonitrile in 60 min. The volume of sample injection was 20 l in all the cases. The peaks of Shatavarin were identified by comparing the retention time of the peaks with those of the reference compounds eluted under same conditions.

7C-EXPECTED RESULTS

Standardization of media for callus induction

Based on the preliminary results, a media composition was standardized for A. racemosus. The details of the combinations used are presented in Table 1. The best combination for callus induction from nodal explants was found to be 1.0 mg/L NAA + 1.0 mg/L 2,4-D + 0.5 mg/L BAP . Preliminary results showed that compared to single auxin, a combination of two auxins is more suitable for the callus induction. A high auxin to cytokinin ratio was

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found to be better suited for induction and proliferation of callus in case of nodal explants. In all the tubes fast growing green compact callus appeared within 25 to 30 days of inoculation. Presence of single auxin 2,4-D alone at high concentrations resulted in a brownish yellow callus (Figure 2b) and a high concentration of NAA alone, resulted in fresh green colored callus with low proliferative capacity. These negative effects were totally absent when these two hormones were used in combination at 1.0 mg/L along with 0.5 mg/L BAP. However a slight increase in the 2,4-D level (1.0 to 1.2 mg/L) in the induction media gives higher proliferative efficiency to the induced callus. Therefore after 2-3 subcultures in the induction medium, calli were maintained in MS media supplemented with 1.0 mg/L NAA + 1.2 mg/L 2,4-D and 0.5 mg/L BAP. Under the above conditions the time required for the induction was about 25 to 30 days.

7D-Studies on induction time

With a view to reduce the induction time required for the callus, the above medium was altered slightly with the addition of extra KH2PO4 (170.0–500.0 mg/L), keeping the hormone combination and levels same. A. racemosus explants were inoculated in the above media. It was noted that induction time decreased to 10 to 15 days when the induction media was supplemented with 250 to 300 mg/L KH2PO4 in case of nodal explants (Table 2). Nodal explants started callusing in around 10 to 15 days, which was nearly half of the previous induction time. This effect on induction time was not seen when another salt of sodium and potassium (K2HPO4) was used. In presence of K2HPO4 explants did not show callus induction either.

7E- Analysis of shatavarin

Callus cultures derived from the nodal and root explants were screened for the presence and accumulation of saponins at various growth phases. The results have been graphically represented in Figure 3. It was found that a callus culture derived from the root explants produces more saponin compared to nodal callus cultures and maximum accumulation was found to be 10.38 ± 0.14 mg/g after 60 days of inoculation. Total saponins from the nodal calli were found to be 7.69 ± 0.136 mg/g of callus. In the wild type roots Shatavarin IV is generally found to be 0.05 to 0.08% where as in our cultures it was found to be 1.1% which indicates that there is approximately 20 fold increase in the saponin content in the in vitro cultures. The high performance liquid chromatography (HPLC) chromatogram of the in vitro culture and the natural plant root extract were compared and it was found that all the major peaks were present in the in vitro extract and the overall saponin profile was similar to the natural root extract .

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7F-DISCUSSION

The results of the experiments show that A. racemosus can be cultured rather easily in synthetic media, which is generally not the case for most of the monocots. Previous workers have observed callus induction to occur when equal levels of auxin and cytokinin are provided in the medium but we found that a high auxin to cytokinin ratio gives better results for callus induction and morphology for all the explant types. Compared to single auxin in high concentration a combination of auxins is better suited for the induction and growth of callus. The auxin NAA seems to support the development of chloroplast as in presence of NAA, callus acquires green colour. For root explants, a medium supplemented with high levels of auxin is good for induction, but for growth, calli must be transferred to a high cytokinin medium. Many plants are known to behave differently on a high phosphorus medium. Phosphorus in general is believed to support good growth and differentiation and the same is true for potassium too. Some of the earlier reports have even suggested that presence of exogenous additional phosphorus and potassium in terms of KH2PO4 enhances secondary metabolite production, especially hecogenin which is also a steroidal saponin . In A. racemosus, callus induction time was reduced to half when the explants were grown on a medium with high auxin, low cytokinin and high KH2PO4. Therefore this can be considered as a good variation if fast induction in the explants is the objective and may also serve to enhance saponin production. The above results prove that in vitro cultures can be used as an alternative source for Shatavarins. However, further work need to be done on establishment of suspension cultures, secretion of saponins into liquid media and evaluation of bioactivities to validate the protocol for scale up studies.

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8-Asparagus Racemosus propagation methods through tissue culture technique

8A-Explant Source:  Shoots from 2 years old plants of A. racemosus grown in the medicinal plant nursery, Lovely Professional University.

8B-Plant cell culture

Plant cell culture is based on the unique property of the cell-totipotency. Cell-Totipotenci is the ability of the plant cell to regenerate into whole plant. This property of the plant cells has been

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exploited to regenerate plant cells under the laboratory conditions using artificial nutrient mediums. With the advances made in genetic engineering, it became possible to introduce foreign genes into cell and tissue culture systems. This led to the development of Genetically Modified (GM) Or Transgenic Crops which had improved traits and characteristics.

8C-Maintenance of Aseptic Environment It is very important to maintain aseptic environment during the in vitro culture of plant cells and tissues. Following are some of the methods adopted for sterilization:(a) Sterilization of Glassware- The glassware can be sterilized in a hot air oven at 160-1800C for 2-4 hours.(b) Sterilization of instruments- The metallic instruments are incinerated by dipping them in 75% ethanol followed by flaming and cooling.(c) Sterilization of nutrient media- The culture media are transferred into glass container, plugged with cotton or sealed with plastic closures and sterilized by autoclaving at 15 psi for 30 min. The autoclaving denatures the vitamins, plant extracts, amino acids and hormones therefore the solution of these compounds are sterilized by using Millipore filter paper with pore size of 0.2 micrometer diameter.(d) Sterilization of plant materials- The surface of the plant material is made sterile by using disinfectants e.g. sodium hypochlorite, hydrogen peroxide, mercuric chloride, or ethanol. The transfer of sterile plant material on to the nutrient medium is done under the cabinet of laminar airflow.(e) Sterilization of Culture room and transfer area- the floor and walls of the culture room should be washed with detergent followed by 2% sodium hypochlorite or 95% ethanol. The sterilization can also be done by exposure to UV light. The cabinet of laminar air flow is sterilized by exposing to UV light for 30 min. and 95% ethanol 15 minutes before starting the work.

8D-Murashige-Skoog Medium Composition (Table 1)

Ingredient mg/L

NH4NO3 1650

KNO3 1900

CaCl2•2H2O 440

MgSO4•7H2O 370

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KH2PO4 170

H3BO3 6.2

MnSO4•H2O 16.9

ZnSO4•H2O 8.6

KI 0.83

NaMoO4•2 H2O 0.25

CuSO4•5H2O 0.025

CoCl2•6H2O 0.025

FeSO4•7H2O 27.8

Na2EDTA•2H2O 37.3

myo-inositol 100

nicotinic acid 0.5

thiamine•HCl 0.1

pyridoxine•HCl 0.5

glycine 2.0

Sucrose 30000

Agar 8000

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8E-Culture Medium :

For all the experiments MS medium was used. The constituents given in Table 1,  were dissolved in double distilled water and the different stock solutions were made. The volume of stock solution was maintained as per requirements of the experiments. The stock solutions were stored in the refrigerator and used with in a month of their preparation. Stocks of growth regulators were added to the medium before autoclaving as per need of the experiments. All auxins were dissolved in few drops of absolute alcohol and cytokinin in few drops of 1 N NaOH and made into required volume. 3% sucrose was added in each combination as carbon source and 1.0% agar was used for solidification of the culture medium. pH of the medium was adjusted to 5.8 by adding 1 N HCl or 1 N NaOH before autoclaving the medium. The medium is then transferred to Erlenmeyer conical flasks (100 ml) and culture tubes (250x15 mm) of Borosil. Approximately 40 ml and 20 ml medium were dispensed in each flask and tube respectively, and ten replicates were taken for each set of experiment. The medium was sterilized in an autoclave at 15 psi for 20 min.

8F-Surface sterilization of explants :

The explants collected from nursery of Botany Department, were washed thoroughly with tap water and were kept under running tap water for 30 min, then surface sterilized with 70% alcohol for 3 min followed by 0.1% HgCl2 with a few drops of liquid detergent for 2-5 min depending upon the plant material. These were subsequently washed 4-5 times with sterilized double distilled water to remove the traces of sterilant. At last, suitable explants were selected and were transferred to in sterilized petriplate for inoculation in culture vessels.

8G-Inoculation and Incubation :

Explants were inoculated horizontally on to medium in culture vessels with each culture vessel containing single explant. All cultures were incubated at 25±2 oC at 50-60% RH under cool white fluorescent light at 30 mE.m-2.s-1 with a 16 h photoperiod. Temperature, RH and light conditions were manipulated according to experimental conditions to get optimal results.

8H-Regeneration was carried out in following stages

8H-a-Induction of multiple shoot buds and callus :

For induction of shoot buds and callus, desired explants were inoculated on the medium containing various levels of growth regulators or their growth adjuvants. Initially cultures were examined every day upto a week of inoculation, later weekly and finally morphogenic responses were recorded after 30-40 days.

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8H-b-Proliferation of multiple shoot buds and callus :

Multiple shoot buds were sub cultured after 40 days on fresh media once and then on medium containing various concentrations of auxins and cytokinis. Callus (with or without shoot bud initials) obtained during primary culture was transferred to fresh medium after 4-5 weeks of culture in order to evaluate whether, callus showed any morphogenic response or continued to proliferate as such. Renewable callus was stocked by subculturing the same on the callus induction medium for further experimentation.The regenerable callus was also further subjected to shoot induction medium. Once the shoot buds were initiated, the same was further sub cultured in order to have shoot formation. Thus, large number of proliferated shoots was obtained.

8I-Hardening of plantlets and pot transfer :

All the plantlets thus obtained in vitro from axillary buds, shoot tips or from callus were carefully removed from culture vessels. They were thoroughly washed with water to remove the traces of media and agar, hardened and then transfer to soil.

9- Pharmacological applications of Asparagus Racemosus

Asparagus Racemosus has been used in Ayurveda as a galactagogue, aphrodisiac, anodyne, diuretic, antispasmodic and nervine tonic since time immemorial. The plant finds use in about 64 ayurvedic formula-tions which include traditional formulations such as ‘Shatavari kalpa’, ‘Phalaghrita’, ‘Vishnu taila’, etc.. Abana® (containing 10 mg Satavari root extract per tablet), Diabecon®(containing 20 mg Satavari root extract per tablet), EveCare®(containing 32 mg Satavari root extract per 5 ml syrup), Geriforte®(containing 20 mg Satavari root powder per tablet), Himplasia®(containing 80 mg Satavari root powder per tablet), Lukol®(containing 40 mg Satavari root extract per tablet) and Menosan®(containing 110 mg Satavari root extract per tablet) are some formulations containing Asparagus Racemosus developed by Himalaya Herbal Healthcare, India (Table 1).

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Fig. 1. Active principles of Asparagus Racemosus (I) Shatavarin, (II) Sarsasa-pogenin, (III) Racemosol and (IV) Asparagamine.

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9A-Effect on neurodegenerative disorders

In Alzheimer’s and Parkinson’s diseases, excitotoxicity and oxidative stress are the major mechanisms of neuronal cell death. Therefore, to combat neurodegenerative disorders, there is a need for a compound that can retard or reverse this neuronal damage. Asparagus Racemosus is a well-known nervine tonic in the Ayurvedic system of medicine. Parihar and Hemnani (2004) conducted a study to investigate the potential of methanolic extract of Asparagus Racemosus roots against kainic acid (KA)-induced hippocampal and striatal neuronal damage in mice. Intra-hippocampal and intra-striatal injections of KA to anes-thetized mice resulted in the production of excitotoxic lesions in the brain. After KA injection, impairment of hippocampus and striatal regions of brain was observed accompanied by increased lipid peroxidation, increased protein carbonyl content, decreased glutathione peroxidase (GPx) activity and reduced glutathione (GSH) content. GSH is an important antioxidant which acts as a nucleophilic scavenger of toxic compounds and as a substrate in the GPx-mediated destruction of hydroperox-ides which would otherwise accumulate to toxic levels in brain tissues. The mice treated with Asparagus Racemosus extract showed an enhancement in GPx activity and GSH content, and reduction in membranal lipid peroxidation and protein carbonyl. They concluded that the plant extract plays the role of an antiox-idant by attenuating free radical induced oxidative damage. ‘EuMil’, a polyherbal formulation containing the stan-dardized extracts of Withania somnifera , Ocimum sanctum , Asparagus Racemosus and Emblica officinalis was evaluated for its anti-stress activity in rats (Bhattacharya et al., 2002). Chronic electroshock stress for 14 days was found to increase the rat brain tribulin activity and decrease the monoamine neuro-transmitter levels. ‘EuMil’ treatment normalized the perturbed nor-adrenalin, dopamine and 5-hydroxytryptamine concentra-tions and also attenuated the tribulin activity. ‘Mentat’, a herbal psychotropic preparation containing Asparagus Racemosus has been found to be effective in the treat-ment of alcohol abstinence induced withdrawal symptoms such as tremors, convulsions, hallucinations and anxiety in ethanol administered rats (Kulkarni and Verma, 1993) due to its anticon-vulsant and anxiogenic action. However, it is unlikely that these are the only reasons for its de-addiction potential and therefore can be examined further. Neurological and psychiatric disorders together account for more chronic suffering than all other disorders combined (Cowan and Kandel, 2001). Treating these problems however, remains a challenging field in medical science. Keeping in mind the encouraging leads and the limited data regarding the use of Asparagus Racemosus in treating neurological disorders; more studies need to be conducted to fully exploit the potential of Satavari in this area.

9B-Anti-diarrhoeal effects

Diarrhoea has long been recognized as one of the most important health problems faced globally particularly by the population of developing countries. Each year diarrhoea is esti-mated to kill about 2.2 million people globally, a majority of whom are infants and children below the age of 5 years (WHO, 2005). Nanal et al. (1974) found Satavari to be extremely effective in the

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treatment of Atisar (diarrhoea), Pravahika (dysentery) and Pittaj shool (gastritis) as described in Ayurvedic texts such as Sushruta Samhita and Sharangdhar Samhita . Ethanol and aque-ous extracts of Asparagus Racemosus roots exhibited significant anti-diarrhoeal activity against castor oil induced diarrhoea in rats demonstrating an activity similar to loperamide .The release of ricinoleic acid from castor oil results in inflammation and irritation of the intestinal mucosa caus-ing the release of prostaglandins which stimulate motility and secretion. It is well known that ‘prostaglandin E’ causes diar-rhoea in experimental animals and human beings. Therefore, the action of this extract can be attributed to the inhibition of prostaglandin biosynthesis which in turn inhibits gastro-intestinal motility and secretion. Since the Asparagus Racemosus root extract is composed of saponins, alkaloids, flavonoids, sterols and terpenes; further analysis is needed to identify the exact phytoconstituent(s) that imparts the anti-diarrhoeal action.

9D-Anti-dyspepsia effects

Asparagus Racemosus also finds use in Ayurveda in the treat-ment of dyspepsia. The plant was found to have an effect comparable to a modern allopathic drug metoclopramide which is a dopamine antagonist used in dyspepsia to reduce gastric emptying time. In this study, 2 g powdered root of Asparagus Racemosus was compared to a standard treatment of metoclopramide (10 mg tablet) in eight normal healthy male vol-unteers, and the gastric emptying halftime was observed. There was no statistically significant difference between the actions of Asparagus Racemosus and metoclopramide. They hypoth-esized that Satavari might be a mild dopamine agonist. This isolated study merely supports the use of Satavari in traditional Ayurvedic medicine as an anti-dyspeptic drug. It does not elabo-rate its mechanism of action which can be an avenue for further research.

9E-Cardio protective effects

Increase in serum lipid levels especially cholesterol along with the generation of reactive oxygen species are the major reasons for the development of coronary artery disease and atherosclerosis. ‘Abana’, a herbo-mineral formulation contain-ing 10 mg Asparagus Racemosus extract per tablet, was found to have significant hypocholesterolaemic effect in rats and there-fore demonstrated a potential for use as a cardio-protective agent . They found that the total choles-terol, phospholipids and triglyceride levels were significantly lower (37–45%) as against the control. Since ‘Abana’ is a poly-herbal formulation, further research needs to be conducted on the exact role that the Asparagus Racemosus component plays in the hypolipidaemic action. Asparagus Racemosus has also been investigated for the reduction of cholesterol levels in hypercholesteremic rats by Visavadiya and Narasimhacharya (2005). They found that Asparagus Racemosus root powder supplements decreased lipid peroxidation and caused a dose-dependent reduction in lipid profiles. The total lipids, total cholesterol and triglycerides in plasma and liver as well as plasma LDL (low-density lipopro-tein) and VLDL (very low-density lipoprotein)-cholesterol decreased by more than 30%. Though it can be hypothesized that the

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hypercholesteremia is alleviated by decreasing exogenous cholesterol absorption and increasing conversion of endogenous cholesterol to bile acid; more research needs to be conducted to comprehend the mechanism of action responsible for this action.

9F-Anti-bacterial effects

In an isolated study, different concentrations of the methanol extract of the roots of Asparagus Racemosus have also shown considerable antibacterial efficacy under in vitro con-ditions against Escherichia coli , Shigella dysenteriae , Shigella sonnei, Shigella flexneri , Vibrio cholerae , Salmonella typhi , Salmonella typhimurium , Pseudomonas putida , Bacillus sub-tilis and Staphylococcus aureus (Mandal et al., 2000b). The antibacterial effect of Asparagus Racemosus may also be play-ing a secondary role in its action with respect to other functions of the plant as well and therefore needs to be studied in greater detail.

9G-Immunoadjuvant effects

The immunoadjuvant potential of Asparagus Racemosus was studied in experimental animals immunized with diphtheria, tetanus, and pertussis (DTP) vaccine. After challenge, animals treated daily with Asparagus racemo-sus aqueous root extract (100 mg/kg body weight) showed a significant increase (p = 0.0052) in antibody titres to Bordetella pertussis as against the untreated animals. Reduced mortality coupled with overall improved health status was observed in treated animals and this indicated the development of a protec-tive immune response. Extracts and formulations prepared from Asparagus race-mosus exhibited various immunopharmacological actions such as increases in white cell counts, haemagglutinating and haemolytic antibody titres in cyclophosphamide (CP)-treated mouse ascitic. CP is widely used in the treatment of a variety of malignant and non-malignant immunopathological disorders and has several side effects such as leucopenia, anaemia, etc. Since macrophages play an important role in the development of intraperitoneal adhesions, the modulation of macrophage activity would pro-vide a new approach for the prevention and management of post-operative adhesions. Asparagus Racemosus being reported to be an immunomodulator and immunostimulant, significantly decreased the adhesion scores by increasing macrophage phago-cytosis by more than 50% in experimental animals treated with the plant extract. It was demonstrated that a combination of Asparagus Racemosus , Withania som-nifera and Tinospora cordifolia extracts protected mice against CP induced neutropenia. Pre-treatment for 15 days with these drugs produced a striking leucocytosis with a predominant neu-trophilia. The leucopenia and specifically neutropenia induced by CP was significantly reduced by the plant extract. The total WBC and absolute neutrophil counts following the treatment with Asparagus Racemosus had risen very high (18800 ± 2001, 13366 ± 501.96, respectively) such that the percentage fall after CP administration (63.77% total counts, 58.13% neu-trophil counts) was greater than that in the control group (43.78%, 25.18%, respectively) (Dhuley, 1997). In addition, it was found that Asparagus Racemosus , Withania somnifera and Tinospora cordifolia along with Picrorhiza

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kurrooa significantly inhibited carcinogen ochratoxin-induced suppression of chemo-tactic activity and production of interleukin-1 and TNF-by macrophages (Thatte et al., 1987). The role of Asparagus Racemosus as an immunoadjuvant in traditional therapy is well documented and therefore it can be applied to evade the toxic side effects of synthetic chemothera-peutic drugs without compromising on its anti-tumour activity. Interestingly, in Ayurvedic medicine, AIDS is thought to be a disease of decreased ‘ojas’, defined as the essential energy of the body. Satavari is said to aid in the formation of ‘ojas’ and has been used in immune therapy (Canadian AIDS Treatment N. Bopana, S. Saxena / Journal of Ethnopharmacology 110 (2007) 1–15 11 Information Exchange, 2005). It is in situations like these that the function of Asparagus Racemosus as an immunoadjuvant can be scrutinized for use in adjuvant therapy in the management of HIV.

9H-Antitussive effects

In yet another isolated report the methanol extract of Aspara-gus racemosus roots showed significant antitussive activity on sulphur dioxide induced cough in mice with the cough inhibition being comparable to that of 10–20 mg/kg of codeine phosphate (Mandal et al., 2000a). This action has not been well documented and can be worked upon further.

10-Challenges in conservation and sustainable use of Asparagus Racemosus

Due to its multiple uses, the demand for Asparagus racemo-sus is constantly on the rise; however the supply is rather erratic and inadequate. Destructive harvesting combined with habitat destruction in the form of deforestation adds to the magnitude of the problem. All of this has resulted in the drastic shrinkage of its population. In nature, the species is propagated through seeds in March–April. Apart from this method, Aspara-gus racemosus can also be propagated vegetatively but this is a very slow and laborious technique. Irrespective of the mode of propagation the plant is ready for harvesting only by the third year. Hence, this is not an effective solution to meet the growing demand for this plant. Considering the escalating demands of the market for a continuous and uniform supply of the plant material, and the increasing depletion of the forest resource base, cultivation of the plant rather than collection from wild will be an effective strategy. However, the basis for a sound conservation strategy would lie not only in increasing the area under cultivation, but also in attaining greater productivity to ensure reasonably higher financial returns to the growers.

11-Cultivation practices

Cultivation has several advantages over collection from the wild. Due to genetic and environmental differences, wild har-vested plants normally vary in quality and consistency which seriously compromises economic returns. Local environmental conditions strongly influence the effi-cacy of medicinal plants. A plant may grow well under different conditions but

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might fail to produce the active constituents of interest since temperature, rainfall, day length and soil character-istics are some of the factors that affect the potency of medicinal plants. These are some parameters that need to be studied well before arriving at suitable agro-techniques

11A-Organic cultivation

Superior quality planting material produced through micropropagation in conjunction with use of bio-fertilizers (mycorrhiza), biopesticides and improved agro-techniques would augment the yields even further and generate consistently higher financial returns to the growers. In addition, since plants propagated by organic farming are preferred in the market, this would also encourage cultivation which would in the long run complement the conservation process.

11B-Post-harvest handling

The quality of medicinal plants depends on the geographi-cal origin, time and stage of growth when collection has been made and the manner of post-harvest handling. In India, vil-lagers who are the major collectors of herbal material pay little attention to quality during harvesting, handling and stor-age. It has been reported that stored herbal drug samples very often harbour mycotoxin-producing fungi. Detection of mycotoxins has been reported in stored roots and rhizomes of Asparagus Racemosus to the tune of 0.16 g/g plant material. Such herbal drugs containing mycotoxins above the accept-able limit fixed by the WHO for human consumption would be rejected in the global market . Efforts should therefore be made to promote sustainable management of medicinal plants at the community level itself by emphasiz-ing on the improvement of collection, cultivation and marketing practices. The trade and economics of Asparagus Racemosus is affected by the presence of adulterants such as roots of Asparagus offici-nalis and Asparagus lucidus (syn. Asparagus cochinchinensis ). According to the British Pharmacopoeia, the substance-specific requirements for Asparagus Racemosus dried roots include, less than 10% loss on drying, less than 1% foreign matter, less than 5% total ash, less than 0.6% acid-insoluble ash, more than 40% ethanol soluble extractive (45% ethanol), more than 34% water soluble extractive, less than 10 ppm total heavy metals and less than 20 ppb aflatoxin (Australian Government Department of Health and Ageing). Consistency in quality and biological activ-ity is crucial in ensuring the efficacy and safety of any botanical drug. Phytochemical investigations are beneficial for standard-ization and dose determination of herbal drugs. DNA based molecular marker techniques can be used in the identification of authentic Asparagus Racemosus material and in the detection of adulterants in the herbal material as has been done in plants like Artemisia annua (Joshi et al., 2004). Fig. 2 illustrates the complete strategy for the conservation and sustainable utilization of Asparagus Racemosus .

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Fig. 2. Multifaceted strategy for the conservation and sustainable utilization of Asparagus racemosus.

12 -Uses & Benefits of Asparagus

I. The roots are useful in nervous disorders, dyspepsia, tumors, scalding of urine, throat infections, tuberculosis, cough bronchitis and general debility.

II. It helps with nervousness, pain, restless sleep, disturbing dreams and people with weak emotional and physical heart.

III. The herb is useful for treating anorexia, insomnia, hyperactive children and people who are under-weight.

IV. Asparagus is considered as a rejuvenating female tonic for overall health and vitality. V. Satawari is used for treating sexual debility, infertility in both the sexes, and menopausal

symptoms and increases milk secretion during lactation. VI. The herb is useful in pregnancy for threatened abortion.

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VII. It is useful for the treatment of ulcerative disorders of stomach and Parinama Sula, clinical entity akin to the duodenal ulcer diseases.

VIII. The paste of fresh leaves is used to apply on the burning sensation of the skin in smallpox and bullae.

IX. The fresh juice of the roots, mixed with honey, helps in reducing the burning sensation pain in tumors, due to pitta.

X. Asparagus proves to be an effective demulcent for the dry and inflamed membranes of the lungs, stomach, kidneys and sexual organs.

XI. The herb is also an extremely nutritious tonic for women from menarche to menopause. XII. Since it increases the urinary output, it is beneficial in urinary stones and Dysurea.

XIII. Asparagus is anabolic to uterus and thus, helpful in uterine hypoplasia in young girls. XIV. It improves uterine growth, mitigates dysmenorrheal and menorrhagia, augments fertility

and imparts anabolic properties. XV. The herb works as a rejuvenative to improve eyesight, when consumed for a prolonged

duration. XVI. When taken with a cup of saffron milk, asparagus is good for post-menopausal women.

XVII. It curbs the intensity of the bronchospasms and decreases the frequency of paroxysms.

13-Qualitative phytochemical analysis The hydro-alcoholic and aqueous extracts of Asparagus racemosus were subjected to different chemical tests for the detection of phytoconstituents such as carbohydrates, glycosides, alkaloids, proteins, amino acids, tannins, phenolics, saponins, flavonoids, triterpenoids, steroids, fixed oils, gums and mucilages.

13a-Tests for carbohydratesThe carbohydrates were tested by using Benedict's test, Fehling's test, Molisch test and Barfoed's test.

13b-Tests for alkaloids The alkaloids have been tested by using Dragendroff's test, Wagner's test, Mayer's test and Hager's test.

13c-Tests for proteins and amino acidsTests like Biuret test, Xanthoprotein test, Lead Acetate test and Ninhydrin test were used for the analysis of proteins and amino acids.

13d-Tests for tannins and phenolicsTest for tannins and phenolics were performed by adding 2-3 drops of ferric chloride to 1ml of extract for the formation of a dark blue or greenish black colour product which shows the presence of tannins.

13-eTest for flavonoidsFlavonoids were tested by means of Shinoda Test.

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13f-Test for triterpenoidsTest for triterpenoids was done by dissolving two or three granules of tin metal in 2 ml thionyl chloride solution and then, add 1 ml of the extract into the test tube. The formation of a pink colour indicates the presence of triterpenoids.

13g-Tests for steroidsThe steroids were tested by using Libermann Burchard test, Salkowski test and Liebermann's reaction.

13h-Test for saponinsThe procedure adopted for the identification of saponins was to take 1 ml of extract which is diluted with 20 ml distilled water and then shaken in a graduated cylinder for 15 minutes. A 1 cm layer of foam indicates the presence of saponins.

13i-Tests for fixed oilsThe fixed oils have been tested by means of Spot test and Saponification test.

13j-Tests for glycosidesTests like Legal test, Baijet test, Borntrager's test and Keller Kiliani Test were used for the analysis of glycosides.

13k-Test for gums and mucilagesTest for gums were performed by hydrolyzing the 1 ml of extract using dil. HCl (3ml). Then Fehling’s solution is added drop by drop till red colour is developed. Test for mucilages were carried out by treating 1 ml of extract with 2 ml of ruthenium red solution to get red colour solution.

14-Literature Review

14a-Asparagus Root Regulates Cholesterol Metabolism and Improves

Antioxidant Status in Hypercholesteremic Rats

Hyperlipidemia/hypercholesteremia are major risk factors for atherosclerosis and cardiovasculardiseases. Root of Asparagus Racemosus (AR) is widely used in Ayurvedic system of medicine inIndia and is known for its steroidal saponin content. This study was designed to investigate thehypocholesteremic and antioxidant potential of AR root in both normo- and hypercholesteremicanimals. Normal and hypercholesteremic male albino rats were administered with root powder of AR (5 and 10g% dose levels) along with normal and hypercholesteremic diets, respectively, for a duration of 4 weeks. Plasma and hepatic lipid profiles, fecal sterol, bile acid excretion and hepatic antioxidant activity were assessed. Inclusion of AR root powder in diet, resulted in a dose-dependant reduction in plasma and hepatic lipid profiles, increased fecal excretion of cholesterol, neutral sterol and bile acid along with increases in hepatic HMG-CoA reductase activity and bile acid content in hypercholesteremic rats. Further, AR root also improved the

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hepatic antioxidant status (catalase, SOD and ascorbic acid levels). No significant changes in lipid and antioxidant profiles occurred in the normocholesteremic rats administered with AR root powder. AR root appeared to be useful as a dietary supplement that offers a protection against hyperlipidemia/hypercholesteremia in hypercholesteremic animals. The results of the present study indicate that the potent therapeutic phyto-components present in AR root i.e. phytosterols, saponins, polyphenols, flavonoids and ascorbic acid, could be responsible for increased bile acid production, elimination of excess cholesterol and elevation of hepatic antioxidant status in hypercholesteremic conditions.

14b-EFFECT OF MIXING ANTIBIOTICS WITH ASPARAGUS RACEMOSUS AND THEIR ANTI-BACTERIAL ACTIVITY M.R. PRAJAPATI AND P.J. VYAS SHETH M.N. SCIENCE COLLEGE, PATAN, Published on: 1st March 2011ABSTRACT: Authors report that Asparagus racemosus has an antimicrobial activity against common pathogens, but when it is combined with Antibacterial drug like Roxythromycin, Cefixime and Levofloxacin the combinations help in - inhibiting growth of Staphylococcus aureus, Staphylococcus epidermis, Escherichia coli and Bacillus subtilis. However, these combinations have powerful effect against bacteria with less side- effects. The medicinal importance of the Asparagus racemosus in the prevention of aerobic and anaerobic bacterial infections is obvious considering the growing number of these developing resistance organisms to conventional antibiotics. Phytochemical Analysis of the Asparagus racemosus helps to find out the presence of chemical constituents in the plant extract. In the present paper, the status on the above mentioned combinations has been discussed. KEY WORDS: Aerobic and anaerobic bacteria, Antibacterial activity, Plant extract, Asparagus racemosus , Roxythromycin, Levofloxacin , Cefixime.

14c-Journal of Ethnopharmacology 110 (2007) 1–15ReviewAsparagus racemosus—Ethnopharmacological evaluation and conservation needsBy Nishritha Bopana a,1, Sanjay Saxena

Abstract

Asparagus racemosus is mainly known for its phytoestrogenic properties. With an increasing

realization that hormone replacement therapy with synthetic oestrogens is neither as safe nor as effective as previously envisaged, the interest in plant-derived oestrogens has increased

tremendously making Asparagus racemosus particularly important. The plant has been shown to

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aid in the treatment of neurodegenerative disorders and in alcohol abstinence-induced withdrawal symptoms. In Ayurveda, Asparagus racemosus has been described as a rasayana herb

and has been used extensively as an adaptogen to increase the non-specific resistance of

organisms against a variety of stresses. Besides use in the treatment of diarrhoea and dysentery,

the plant also has potent antioxidant, immunostimulant, anti-dyspepsia and antitussive effects. Due to its multiple uses, the demand for Asparagus racemosus is constantly on the rise; however,

the supply is rather erratic and inadequate. Destructive harvesting, combined with habitat

destruction in the form of deforestation has aggravated the problem. The plant is now considered

‘endangered’ in its natural habitat. Therefore, the need for conservation of this plant is crucial. This article aims to evaluate the biological activities, pharmacological applications and clinical

studies of Asparagus racemosus in an attempt to provide a direction for further research. Keeping in mind the fact that it is the active principle that imparts medicinal value to a plant;

consistency in quality and quantity needs to be maintained to ensure uniform drug efficacy. Also,

deliberate or inadvertent adulteration needs to be dealt with at an early stage. To overcome these prevalent problems, the availability of genetically superior and uniform planting material is

essential. This can be obtained by a combination of various biotechnological tools involving chemoprofiling, tissue culture and use of molecular markers. Along with the application of these

methods, proper agro-techniques and adequate marketing opportunities would encourage

cultivation of Asparagus racemosus and thereby contribute to its conservation. There are also several gaps in the existing literature with regard to the pharmacological actions of Asparagus

racemosus . These include an incomplete understanding about the interaction/synergy between

Asparagus racemosus and other plant constituents in polyherbal formulations; lack of information regarding the mode of action of the various constituents of Asparagus racemosus ,

etc. Consequently, we have suggested a ‘systems biology’ approach that includes metabolite

profiling, metabolic fingerprinting, metabolite target analysis and metabonomics to enable further research.

Keywords: Asparagus racemosus; Conservation; Immunostimulant; Phytoestrogen; Shatavarin

14d-ETHNOPHARMACOLOGY, PHYTOCHEMISTRY AND BIOACTIVITY OF ASPARAGUS RACEMOSUS: AN UPDATE

Correspondence author: Dr. Vandana Garg,

Department of pharmaceutical sciences, M.D. University, Rohtak, India,124001.

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Abstract:

Asparagus racemosus is an herbaceous perennial plant. It is the most important rasayana herb in Ayurvedic medicine which grows in low forest areas throughout India. Their medicinal usages have been reported in indigenous systems of medicine, Indian and British Pharmacopoeias. In Ayurveda, A. racemosus has been used extensively as an adaptogen to increase the non,specific resistance of organisms against a variety of stress. A. racemosus plant contain steroidal saponins, isoflavones, asparagamine and polysaccharides, which play a major role in treatment of diarrhoea and dysentery. Roots and rhizomes of A. racemosus has potent antioxidant, antitussive, antidyspepsia, antiulcer and anticancer activity. A. racemosus is also useful as immunostimulant, galactogogue. The present article gives the detailed exploration of phytochemistry, ethnopharmacology and bioactivity of A. racemosus, in an attempt to give a direction for further research.

Keyword: Asparagus racemosus, Phytochemistry, Galactogogue, Steroidal saponins.

15-Conclusion

The pharmacological studies conducted on Asparagus race-mosus indicate the immense potential of this plant in the treatment of conditions such as menopausal symptoms, neurode-generative disorders, diarrhoea, dyspepsia, etc. However, gaps in the studies conducted are apparent which need to be bridged in order to exploit the full medicinal potential of Asparagus Racemosus . Since most drugs containing Satavari that are available in the market are in the form of polyherbal formulations, it is difficult to attribute a particular medicinal action as being solely due to the Asparagus Racemosus component of the drug. Aside from possible synergistic effects existing among the plant extract constituents, another likelihood could be the formation of ‘pro-drugs’ which are defined as compounds activated in the body after the administration of a particular medicineWhile most of the research has been in vivo which has helped to validate the applicability on the human system; in vitro stud-ies would have facilitated a better understanding of the mode of action of Asparagus Racemosus . Due to the non-availability of commercial Shatavarin standards, most studies first involve the extraction and purification of the active principle to be used as a reference standard which makes the process more cumber-some. The availability of authentic metabolite standards would not only hasten secondary metabolite assays but also make the results more reliable and reproducible. All of these issues can be addressed by adopting a ‘systems biology’ approach wherein a very large number of com-ponents (at the levels of genome, transcriptome, proteome or metabolome as well as physiological parameters such as blood pressure, etc.) are catalogued and statistical methods are used to infer correlations to understand mechanisms of action. A key technology in systems biology is ‘metabolomics’. Metabolomics refers to the exhaustive profiling (identifica-tion, analysis and quantification) of the ‘metabolome’ which includes all the metabolites in a cell. It is a complex inter-disciplinary

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field of research that requires a combination of bioscience, analytical chemistry, organic chemistry, chemomet-rics and informatics. Strategies for metabolomics analysis can include the metabolite profil-ing of Asparagus Racemosus by identification and quantification of pre-defined metabolites by chromatographic separations (by high performance liquid chromatography, gas chromatogra-phy, and electrophoresis) followed by spectroscopic detection (by mass spectrometry or nuclear magnetic-resonance spec-trometry). Metabolic fingerprinting should be done for sample classification purposes to ensure purity and avoid deliberate or inadvertent adulteration with material from other Aspara-gus species.

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Bhatnagar, M., Sisodia, S.S., Bhatnagar, R., 2005. Antiulcer and antioxidant activity of Asparagus racemosus Willd. and Withania somnifera Dunal in rats. Annals of the New York Academy of Sciences 1056, 261–278.

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