1-s2.0-s0278691506002985-main

www.elsevier.com/locate/foodchemtox

Food and Chemical Toxicology 45 (2007) 609–617

Adaptogenic and safety evaluation of seabuckthorn(Hippophae rhamnoides) leaf extract: A dose dependent study

S. Saggu, H.M. Divekar, V. Gupta, R.C. Sawhney, P.K. Banerjee, R. Kumar *

Department of Biochemical Pharmacology, Defence Institute of Physiology and Allied Sciences (DIPAS), Lucknow Road, Timarpur, Delhi 110 054, India

Received 14 December 2005; accepted 16 October 2006

Abstract

The effects of seabuckthorn (Hippophae rhamnoides L., Elaeagnaceae), leaf aqueous extract were examined in rats for its adaptogenicactivity and toxicity. Dose dependent adaptogenic study of extract was carried out at different doses administered orally, 30 min prior tocold (5 �C)–hypoxia (428 mmHg)–restraint (C–H–R) exposure. After sub-acute toxicity studies on 10 and 20 times doses of maximaleffective dose administered for 14 days (single oral dose of 1 g/kg and 2 g/kg once daily) and maximal effective dose administered for30 days (single oral dose of 100 mg/kg once daily), biochemical and hematological parameters were studied in the serum and blood.The maximal effective adaptogenic dose of the extract was 100 mg/kg body weight. No significant changes were observed in organweight/body weight ratios, of any vital organ studied (except liver and kidney in 1 g/kg and 2 g/kg body weight doses, respectively),and biochemical and hematological parameters of the sub-acute drug treated animals in comparison to control rats. In acute toxicitystudy LD50 of the extract was observed to be >10 g/kg when given orally. These results indicate that seabuckthorn leaf aqueous extractpossess potent adaptogenic activity with no toxicity even after sub-acute (30 days) maximal effective dose administration.Crown Copyright � 2006 Published by Elsevier Ltd. All rights reserved.

Keywords: Seabuckthorn; Adaptogen; Hypoxia; Cold; Rectal temperature; Toxicity

1. Introduction

Herbal formulations have been in use for many yearsnot only in Asian countries but also globally for humanwell-being (Brekhman, 1980; Fulder, 1980). The herbalformulations claimed to enhance physical endurance; men-tal functions and non-specific resistance of the body havebeen termed as adaptogens (Brekhman, 1980). During the

0278-6915/$ - see front matter Crown Copyright � 2006 Published by Elseviedoi:10.1016/j.fct.2006.10.008

Abbreviations: C–H–R, cold–hypoxia–restraint; cm, centimeter; EDT-A, ethylene diamine tetra acetic acid; g, gram; HPLC, high performanceliquid chromatography; kg, kilogram; LDH, lactate dehydrogenase; LD50,median lethal dose; MCV, mean corpuscular volume; ll, microlitre; mg,milligram; ml, milliliter; mm, millimeter; min, minute; Na+, sodium; K+,potassium; RBC, red blood cells; Trec, rectal temperature; SE, standarderror; SGOT, serum glutamic oxaloacetate transaminase; SGPT, Serumglutamic pyruvate transaminase; v/v, volume/volume; w/w, weight/weig-ht; w/v, weight/volume; WBC, white blood cells.

* Corresponding author. Tel.: +91 11 23911215; fax: +91 11 23914790.E-mail address: [email protected] (R. Kumar).

stressful situations supplementation of various nutrientsand single and poly-herbal preparation have been shownto increase stress tolerance (Bhargava and Singh, 1981;Grover et al., 1995; Kumar et al., 1996, 1999, 2000,2002). It has been hypothesized that plants growing inadverse climatic conditions of high altitude acquire bio-molecules which help them to sustain in such environmentand supplementation of such plant products to the animalsincrease their performance during exposure to stressfulcold and hypoxic environment (Divekar et al., 1996).

Hippophae rhamnoides L., commonly known as sea-buckthorn (family: Elaeagnaceae) growing in North-WestHimalayas at high altitude, 7000–15,000 feet, is a dwarfto tall (3–15 feet), branched, and thorny nitrogen fixingdeciduous shrub, native to Europe and Asia (Rousi,1971). All parts of the plant are considered to be goodsource of a large number of bioactive substances. Thechemical and phytochemical composition of seabuckthornhas been reviewed and found to vary with the origin,

r Ltd. All rights reserved.

mailto:[email protected]

610 S. Saggu et al. / Food and Chemical Toxicology 45 (2007) 609–617

climate and method of extraction (Beveridge et al., 1999;Zeb, 2004). The ripe fruit has been reported to be a sourceof exceptionally high contents of vitamins (A, C, E, and K),carotenoids, flavonoids, and organic acids. Extracts ofwhole fruit, fruit pulp, pulp oil, and seed oil has beenreported to possess immunomodulatory, anti-oxidant, andanti-bacterial activity (Geetha et al., 2002; Negi et al.,2005) and reported to be useful in treating various diseasessuch as gastric ulcers (Suleyman et al., 2001; Xing et al.,2002), skin disorders (Yang et al., 2000), coronary heartdisease (Eccleston et al., 2002), radiation induced oxidativedamage (Goel et al., 2005), wound healing (Gupta et al.,2005) and thrombosis and platelet aggregation (Chenget al., 2003). The medicinal effects of seabuckthorn havebeen suggested to be due to the presence of high anti-oxidant contents (Beveridge et al., 1999; Eccleston et al., 2002).

Seabuckthorn leaves are rich in flavonoids, tannins, andtriterpenes (Kallio et al., 2002). But only few studies areavailable on the pharmacological effects of seabuckthornleaf, as compared to fruits and seed. Seabuckthorn leavesethyl alcohol (70%) extract was reported to possess anti-oxidant and immunomodulatory (Geetha et al., 2003) andanti-inflammatory activity (Ganju et al., 2005). In a singleavailable study, oral administration of single dose (100 mg/kg body weight) extract of seabuckthorn dry leaves extractedwith 70% ethyl alcohol in a Soxhlet apparatus was found topossess anti-stress activity (Geetha, 2004). However, adetailed dose dependent study of seabuckthorn leaf for itsanti-stress adaptogenic activity remained to be carried out.

The present study was undertaken to evaluate dosedependent adaptogenic activity of seabuckthorn driedleaves aqueous extract in rats using a passive cold (5 �C)–hypoxia (428 mm Hg)–restraint (C–H–R) animal model(Ramachandran et al., 1990). Further, for safety studieson seabuckthorn leaf aqueous extract administration acuteand sub-acute toxicity, if any and effect on biochemical andhematological parameters in the serum and blood of ani-mals were determined.

2. Materials and methods

2.1. Experimental animals

Male inbred Sprague-Dawley strain rats, 12–14 weeks old, weighing150 ± 10 g were used for the study. The animals were maintained undercontrolled environment in the institute’s animal house at 25 ± 1 �C and 12-hlight–dark cycle. The animals were fed standard animal food pellet and waterad libitum. The experiments were performed in accordance with the regula-tions specified by the Institutional Animal Ethical Committee and conformedto national guidelines on the care and use of laboratory animals, India.

2.2. Plant material

Seabuckthorn (Hippophae rhamnoides L.) leaves were collected, indifferent batches, on the two different dates in the month of Septemberfrom hilly regions of western Himalayas, India, where the plant growswidely under natural conditions and always from the same region to ruleout phytochemical variations caused due to regional and climatic condi-tions. The Field Research Laboratory, Leh, where the voucher specimenof the plant material is preserved in the herbarium, carried out the

ethanobotanical identification of the plant. Fresh leaves of seabuckthornwere cleaned and washed thoroughly with water and re-washed with dis-tilled water. Washed fresh leaves were dried under shade in a clean, dustfree environment and crushed using laboratory blender.

2.3. Extract preparation

Aqueous extract of seabuckthorn dried leaves was prepared by coldpercolation method. The powdered dry leaves powder was soaked in dis-tilled water (1:5 w/v) at room temperature (25 ± 1 �C). After 24 h, thesupernatant was decanted and the residue re-soaked in fresh water. Theprocess was repeated four times for complete extraction. To avoid con-tamination, clean and sterile conditions were maintained during theextraction process. The supernatants were pooled, filtered through muslincloth and stored in amber colored bottles and centrifuged at 8000g, 4 �C.The supernatant obtained after centrifugation was frozen at �20 �C andthen lyophilized in a Heto Lyophilizer (HITOSICC, Heto-Holten A/S,Denmark). Lyophilized powder of the seabuckthorn leaf aqueous extractwas stored at �20 �C in an airtight glass tube until further use. The crudeyield of the lyophilized extract was determined gravimetrically by weighingthe dried residues obtained out of known volumes of extract, extracted fromknown weight of dry leaves powder. One gram of dried seabuckthorn leavesproduced 0.137 g of lyophilized seabuckthorn aqueous extract powder.

2.4. Characterization of the extract

To characterize the seabuckthorn dry leaves aqueous extract its HPLCfingerprinting and total phenolic content were estimated.

2.4.1. HPLC fingerprinting

HPLC analysis of the seabuckthorn leaves aqueous extract wasperformed using model AS-3000 spectro system (Thermo Finnigan, USA)equipped with 1000 UV detector. A 20.0 ll sample of 1 mg/ml solution ofthe extract was injected followed by implementation of HPLC gradesolvent, methanol and water (40:60 v/v) at 1 ml/min. Reverse phase C-8column (250 mm · 4.7 mm), particle size 5 lm were used to separate thecomponent at 310 nm absorbance.

2.4.2. Total phenolic content

The total phenolic content was estimated in the seabuckthorn leavesaqueous extract using Folin–Ciocalteu reagent (FCR) based assay(Singleton and Rossi, 1965). To an aliquot (20 ll), taken from a stocksolution (1 mg/ml) of the extract, 80 ll of water and 500 ll of FCR wereadded. The mixture was kept for 5 min at room temperature, and then400 ll of 7.5% sodium carbonate solution was added. The mixture waskept for 30 min and absorbance of the colour developed was recorded at765 nm. Total phenols (mg/g) in the seabuckthorn leaves aqueous extractwere expressed as gallic acid equivalent (GAE), using standard curveprepared from gallic acid (0.1 mg/ml) solution.

2.5. Experimental design

All the experiments were done on healthy overnight fasted rats. In thepresent study two experiments were carried out. In the first experiment adose dependent adaptogenic study of seabuckthorn dry leaf aqueousextracts was carried out in rats using cold (5 �C)–hypoxia (428 mmHg)–restraint (C–H–R) animal model (Ramachandran et al., 1990) to evaluatemaximal effective adaptogenic dose of the extract. In another study acute,sub-acute toxicity of seabuckthorn leaf aqueous extract was determined byevaluating serum biochemical parameters, blood hematology and organweight/body weight ratio of vital organs.

2.6. Dose-dependent adaptogenic studies

In total 66 rats were used for the evaluation of seabuckthorn leafaqueous extract adaptogenic activity, including 12 rats to study multiple

S. Saggu et al. / Food and Chemical Toxicology 45 (2007) 609–617 611

dose effect of the selected maximal effective single dose. The seabuckthornleaf lyophilized powder was dissolved in suitable volume of water toobtain the desired dose on body weight basis (mg/kg) of the animal in0.5 ml volume for its oral administration. A single dose of seabuckthorn(Hippophae rhamnoides) dry leaf aqueous extract was given orally in a0.5 ml volume through a gastric cannula to overnight fasted rats, 30 minprior to C–H–R exposure. Six rats were used for each dose. The differentdoses of the seabuckthorn leaf aqueous extracts used were 3.125, 6.25,12.5, 25, 50, 100, 150 and 200 mg/kg body weight. Six control rats wereadministered an equivalent volume (0.5 ml) of water orally 30 min prior toC–H–R exposure (Ramachandran et al., 1990).

The rats were exposed in a decompression chamber maintained at 5 �Cand a low atmospheric pressure of 428-mmHg pressures equivalent to analtitude of 4572 m. The rats were restrained and rectal probe was inserted2 cm past the rectum and retained there with the help of adhesive plaster.The rectal temperature (Trec) of the rats was monitored once per minute,by using a 16-channel Isothermex Temperature Recorder (ColumbusInstrument, Columbus, OH). When the rats attained a rectal temperature(Trec) of 23 �C, they were taken out of the chamber. The rats were allowedto recover to a normal Trec of 37 �C at normal atmospheric pressure andambient temperature 32 ± 1 �C. A constant room temperature wasmaintained in all experiments as recovery time also depended on theambient temperature. The rats continued to be restrained during recoveryperiod. The time taken to attain Trec 23 �C and its recovery to Trec 37 �Cwere used as a measure of endurance (Ramachandran et al., 1990).

After establishing the maximal effective adaptogenic dose of seabuck-thorn aqueous leaf extract its five doses administration effect, single doseper day, was evaluated using C–H–R animal model to ascertain whetherthe multiple dose administration of the extract has any cumulative adap-togenic activity or not. In total 12 rats were used for multiple dose studies,both drug treated and control groups consisted of six rats in each group.

2.7. Toxicity studies

2.7.1. Acute toxicity

In total 48 male rats fasted overnight were used for acute toxicitystudies. The rats were divided in four groups each group consisting of 12rats. One time single dose of 1, 2, 5 and 10 g/kg of aqueous lyophilizedextract was administered orally to each group of overnight fasted rats.After drug administration the animals were provided with food and waterimmediately and closely observed in their cages for any mortality and signsof severe toxic effects such as hypo-activity, piloerection, anorexia, sali-vation, diarrhea, syncope, muscle cramping, convulsions, if any, for 24 hand further daily for next 14 days. In each group number of rats that died,if any, within the period of study were noted to calculate the LD50 value.

2.7.2. Sub-acute toxicity

In total 36 rats were used for sub-acute toxicity studies divided in threegroups consisting of 12 animals in each group. Group one; rats were givenan oral dose of 1 g/kg body weight (10 times effective dose) in 0.5 mlvolume, once a day for 14 days. Second group of rats were given an oraldose of 2 g/kg body weight (20 times effective dose) in 0.5 ml volume, oncea day for 14 days. Group three rats were given 0.5 ml distilled water, oncea day for 14 days and served as controls. Food and water were freelyavailable to the animals during the experiment. Body weight of the ratswere measured every day and recorded. After 14 days of the experiment sixrats from each group were used for biochemical analysis and another sixrats of each group were used for hematology and organ weight/bodyweight ratios. After 14 days of drug treatment, animals were fastedovernight and for clinical chemistry blood samples were obtained fromorbital sinus (Riley, 1960) using capillary tubes (with and without heparinas per requirement) under mild ether anesthesia. Blood for hematologystudies was collected into tubes containing ethylene-diamine-tetra-aceticacid (EDTA) as an anti-coagulant. After animals were sacrificed, vitalorgans (kidney, liver, spleen, adrenal, testis, heart and lung) were carefullydissected out, cleaned of the adhering connective tissues, blotted andaccurately weighed. Ratio of each organ to body weight was determined.

Another 24 rats were used for sub-acute toxicity, if any, after long-term administration of seabuckthorn aqueous leaf extract maximal effec-tive dose. Twelve rats received daily single maximal effective dose (100 mg/kg body weight) of seabuckthorn aqueous leaf extract, orally in 0.5 mlvolume, for 30 days. Another 12 control rats received 0.5 ml distilled wateronce daily for 30 days. Body weight of the animals was recorded daily. Sixrats from each group were used for biochemical analysis. Another six ratsfrom each group were used for hematology. Organ weight/body weightratios were determined of all the 12 rats in each group. After 30 days ofdrug treatment blood of the animals was obtained from the orbital sinus(Riley, 1960) for biochemical and hematological analysis. Kidney, liver,spleen, adrenal, testis, heart and lung were dissected out, cleaned ofadhered tissues, blotted and weighed for organ weight/body weight ratiodetermination.

2.8. Biochemical assay

Fasting blood glucose was estimated using Ascensia Entrust bloodglucose test strips and blood glucose meter (Bayer Diagnostics India Ltd.,Baroda, Gujarat, India). In whole blood lactate dehydrogenase (LDH)activity was determined (Kornberg, 1969). Serum SGOT (King, 1965),SGPT (Wroblewski and LaDue, 1956), alkaline phosphatase (Lowry et al.,1954), bilirubin (Malloy and Evelyn, 1937), creatinine (Bonsnes andTaussky, 1945), total cholesterol (CHOD-PAP Diagnostic kits, Cat.Number E 98118; Velon Pharmacal. Pvt. Ltd., Mumbai, India), triglycer-ides (GPO-PAP Diagnostic kits, Cat. Number E 96388; Velon Pharmacal.Pvt. Ltd., Mumbai, India), total serum protein (Lowry et al., 1951) andserum electrolytes (Na+, K+) using Stat Profile Phox Plus Blood-gas Ana-lyzer (Nova Biomedical, Waltham, MA 02454-9141, USA) were estimated.

2.9. Hematological study

Hemoglobin and other hematological parameters were determined incontrol and drug treated animals using semi-automated micro cell counterSysmex F-820 (TOA Medical Electronics Corporation Ltd., Kobe, Japan).

2.10. Histology

For histological assessment of organ damage, if any, necropsy wasperformed on all animals at the end of the study. The kidney, liver, spleen,adrenal, testes, heart and lung were taken out and then fixed in 10%neutral buffered formalin solution. After fixation, tissues were routinelyprocessed, embedded in paraffin, cut in microtome setting of 4 lm,mounted on glass slides, stained with hematoxylin and eosin (McManusand Mowry, 1965) and examined by light microscopy.

2.11. Statistical analysis

Statistical significance between control and experimental values wascalculated by Student’s t-tests using Graph Pad Prism 2.01 (Graph PadSoftware Inc.). The results of dose dependent studies and biochemical andhematological and organ weight/body weight values of 1 g and 2 g/kgbody weight dose sub-acute toxicity, were analyzed by ANOVA withcorrection for multiple comparisons using Bonferroni multiple range test,using Graph Pad Prism 2.01. The results are presented as means ± SE. Avalue of P < 0.05 was considered as statistically significant.

3. Results

3.1. Characterization of the seabuckthorn leaves aqueous

extract

The HPLC profile of the extract has been given in Fig. 1.The retention time and major peak area were within 2%

0 5 10 15 20 25 30 35 40

0

2

4

6

8

10

mAU

Minutes

Fig. 1. HPLC fingerprinting of the seabuckthorn dry leaf aqueous extract at 310 nm.


variation when analyzed for different batches of the pre-pared extracts.

3.1.1. Total phenolic content

Total phenolic content in the seabuckthorn aqueousleaves extract was found to be 363 mg/g (w/w) in termsof gallic acid equivalent.

3.2. Dose dependent adaptogenic studies

The results on dose dependent studies have been given inTable 1. The lowest studied dose (3.125 mg/kg) of seabuck-thorn aqueous extract was effective only in significantlyfastening the post stress recovery as evidenced by 22.9%less time taken by the rats to recover back Trec 37 �C fromC–H–R induced hypothermia (Trec 23 �C). But the sea-buckthorn aqueous extract up to a dose of 6.25 mg/kghad no effect in providing resistance to C–H–R inducedhypothermia (Trec 23 �C). With the increase in used drugdoses both the resistance to C–H–R induced hypothermiaand faster recovery (Trec 37 �C) from hypothermiaimproved. Administration of 100 mg/kg drug dose signifi-

Table 1Dose dependent adaptogenic activity of seabuckthorn leaf aqueous extract aft

Dose (mg/kgbody weight)

Time taken (in min)to attain Trec 23 �C

% Change fromcontrol values

Control rats 75.5± 0.63.125 73.5 ± 1.7 2.66.25 75.2 ± 1.1 0.412.5 92.5 ± 1.9* 22.525 102.7 ± 1.04* 36.050 104.8 ± 1.1* 38.8100 125.7 ± 1.4* 66.4150 122.8 ± 2.0* 62.6200 107 ± 2.2* 41.7

Results are means ± SE of six rats in each group.* Significance at p < 0.05.

cantly provided maximum resistance to C–H–R inducedhypothermia (66.4%) and also fastened the recovery by42%. However, further increases in drug doses could notprovide more improvement in resistance to C–H–Rinduced hypothermia as well as recovery from hypother-mia. The maximal effective dose of seabuckthorn aqueousleaf extract was observed to be 100 mg/kg body weight.

The result on the multiple dose administration, singledose per day for five days, of seabuckthorn aqueous leafextract have been shown in Table 2. After five doses oraladministration of seabuckthorn leaves aqueous extract(100.0 mg/kg), single dose per day, time taken to attainto Trec 23 �C during exposure to C–H–R and recovery (Trec

37 �C) from C–H–R induced hypothermia, were not differ-ent from single dose (100 mg/kg) effect.

3.3. Toxicity studies

3.3.1. Acute toxicity

The results on acute toxicity studies have been given inTable 3. In none of the four animal groups, treated withone time seabuckthorn leaves aqueous extract at oral doses

er single dose oral administration



157.0 ± 1.5121.2 ± 1.2* 22.999.8 ± 1.69* 36.498.5 ± 3.9* 37.399.5 ± 1.7* 36.698.8 ± 3.7* 37.191.0 ± 3.1* 42.098.2 ± 3.6* 37.5

92 ± 1.4* 41.4

Table 2Adaptogenic activity of seabuckthorn leaf aqueous extract (100 mg/kg) after multiple oral dose administration (single dose/day for 5 days)

Dose (mg/kgbody weight)





Control rats 75.5 ± 0.6 157.0 ± 1.5Single dose 125.7 ± 1.4* 66.4 91.0 ± 3.1* 42.0Five dose 125.5 ± 1.5* 66.2 82.7 ± 1.6* 47.3

Results are means ± SE of six rats in each group.* Significance at p < 0.05.

Table 3Acute toxicity (LD50) of seabuckthorn leaf aqueous extract administeredorally to rats

Dose (g/kgbody weight)

D/T Mortalitylatency (h)

Toxicitysigns

1 0/12 – None2 g/kg 0/12 – None5 g/kg 1/12 >24 None10 g/kg 1/12 >24 None

D/T, dead/treated rats; None, no toxic symptoms during the observedperiod; Mortality latency, time to death (in h) after the oral administrationof the extract; rats in each dose group (n) = 12.

Table 4Effects of sub-chronic seabuckthorn leaf aqueous extract, oral adminis-tration (1 g/kg and 2 g/kg body weight, single dose/day for 14 days), onthe biochemical and hematological parameters

Parameters Control 1 g/kg 2 g/kg

Cholesterol (mg/dl) 94.3 ± 3.4 92.2 ± 2.8 96.7 ± 4.2Triglycerides (mg/dl) 65.9 ± 5.1 62.5 ± 3.6 58.1 ± 4.0Creatinine (mg/dl) 0.5 ± 0.1 0.4 ± 0.03 0.4 ± 0.1Bilirubin (mg/dl) 0.4 ± 0.1 0.3 ± 0.1 0.2 ± 0.04SGPT (IU) 6.7 ± 0.9 5.2 ± 0.9 5.7 ± 0.8Protein (g/dl) 9.3 ± 0.1 8.4 ± 0.7 8.5 ± 0.5WBC (·103 l/l) 4.7 ± 0.7 5.1 ± 0.8 5.7 ± 0.3RBC (·106 l/l) 7.5 ± 0.2 7.1 ± 0.2 8.5 ± 0.2*

Hemoglobin (g %) 14.6 ± 0.3 14.6 ± 0.5 15.7 ± 0.4Hematocrit (%) 44.6 ± 0.7 45.0 ± 1.0 49.5 ± 0.6*

MCV (fQ) 59.8 ± 0.9 61.8 ± 0.8 58.5 ± 1.6Platelets (·103 l/l) 873.8 ± 64.4 865.0 ± 80.8 995.7 ± 48.3


of 1, 2, 5 and 10 g/kg, 50% rats died within 24 h of treat-ment and even after 14 days of drug treatment.

All the values are means ± SE of six rats in each group.* Significance at p < 0.05.

3.3.2. Sub-acute toxicity

All the animals orally administered 1 and 2 g/kg bodyweight dose for 14 days continued to remain active andhealthy throughout the period of study. The treated andcontrol animals gained body weight well in comparisonone another and control animals (Fig. 2). The results onthe biochemical and hematological parameters of animalsorally treated with 1 and 2 g/kg drug dose, once daily for14 days, have been given in Table 4. There was no changein any biochemical parameter studied of the experimentalanimals, administered both 1 and 2 g/kg body weight dosefor 14 days, in comparison to control animals. However, inhematological parameters studied in animals administered2 g/kg body weight drug dose in comparison to controls,there was a significant increase in red blood cell (RBC)

150

200

250

0 2 4 6 8 10 12 14Days after treatment

Bod

y w

eigh

t (g)

Control

1g/kg2g/kg

Fig. 2. Average body weight on alternate days of rats administered 1 gand 2 g/kg dose of seabuckthorn leaf aqueous extract (single dose per day)for 14 days. All the values are means ± SE of 12 rats in each group.

numbers resulting into increased hematocrit value. Theorgan weight/body weight ratio of animals treated with 1and 2 g/kg body weight drug dose are given in Table 5.In 1 g/kg drug dose treated animals except liver all otherorgans (heart, kidney, spleen, adrenal, testis and Lung)showed no significant change in the organ weight/bodyweight ratios. There was a significant increase in liverweight/body weight ratio of animals, in comparison to con-trols. In animals treated with 2 g/kg drug there was adecrease in kidney weight/body weight ratio only whileall other organ (testis, heart, liver, spleen, adrenal andLung) showed unaltered organ weight/body ratios andwere comparable to controls.

Table 5Effects of sub-chronic seabuckthorn leaf aqueous extract oral administra-tion (1 g/kg and 2 g/kg body weight, single dose/day for 14 days) on theorgan weight/body weight ratio of rats

Organ Control 1 g/kg 2 g/kg

Liver · 10�3 30.7 ± 0.7 36.1 ± 1.5* 28.5 ± 0.8Heart · 10�3 3.6 ± 0.04 3.5 ± 0.1 3.4 ± 0.1Kidney · 10�3 4.1 ± 0.04 4.1 ± 0.1 3.7 ± 0.1*

Spleen · 10�3 2.3 ± 0.1 2.3 ± 0.1 2.1 ± 0.1Testis · 10�3 4.2 ± 0.3 4.8 ± 0.4 4.1 ± 0.8Adrenal · 10�5 9.6 ± 0.4 9.6 ± 0.4 10.5 ± 0.5Lung · 10�3 5.5 ± 0.1 5.6 ± 0.1 5.7 ± 0.4


Table 7Effects of chronic seabuckthorn leaf aqueous extract oral administration(100 mg/kg body weight, single dose/day for 30 days), on the organweight/body weight ratio of rats

Organ Control 100 mg/kg

Liver · 10�3 31.3 ± 1.0 31.3 ± 0.7Heart · 10�3 3.0 ± 0.1 3.0 ± 0.1Kidney · 10�3 3.4 ± 0.03 3.5 ± 0.1Spleen · 10�3 1.8 ± 0.04 1.8 ± 0.02Testis · 10�3 4.6 ± 0.1 4.5 ± 0.1Adrenal · 10�5 7.6 ± 0.3 7.6 ± 0.2Lung · 10�3 4.03 ± 0.12 4.20 ± 0.05

All the values are means ± SE of 12 rats in each group.


In sub-acute toxicity of the animals orally administered100 mg/kg body weight dose for 30 days continued toremain active and healthy throughout the period of study.The treated and control animals gained body weight well incomparison to one another and control animals (Fig. 3).The results on the biochemical and hematological parame-ters of animals orally treated with 100 mg/kg maximaleffective adaptogenic dose, once daily for 30 days, havebeen given in Table 6. There was no change in any bio-chemical parameter studied of experimental animals,except a decrease in the serum sodium levels, in comparisonto control animals. In hematological parameters studiedthere was a significant increase in hematocrit and plateletcounts of drug treated animals in comparison to controlanimals. The organ weight/body weight ratio of animals

158.00

208.00

258.00

308.00

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Days after treatment

Bod

y w

eigh

t (g)

Control

100 mg/kg

Fig. 3. Average body weight on alternate days of rats administeredeffective dose (100 mg/kg body weight) of seabuckthorn leaf aqueousextract (single dose per day) for 30 days. All the values are means ± SE of12 rats in each group.

Table 6Effects of chronic seabuckthorn leaf aqueous extract oral administration(100 mg/kg body weight, single dose/day for 30 days), on the biochemicaland hematological parameters of rats

Parameters Control 100 mg/kg

Cholesterol (mg/dl) 92.9 ± 3.2 91.5 ± 4.6Triglycerides (mg/dl) 63.7 ± 4.8 63.2 ± 4.2Creatinine (mg/dl) 0.5 ± 0.1 0.4 ± 0.04Bilirubin (mg/dl) 0.3 ± 0.1 0.3 ± 0.1SGPT (IU) 8.6 ± 2.1 8.3 ± 1.1SGOT (IU) 27.7 ± 2.1 27.0 ± 2.3Alkaline phosphate (IU) 9.4 ± 0.4 9.8 ± 0.4LDH (n moles/mg protein) 10.4 ± 0.4 11.1 ± 0.6Protein (g/dl) 9.4 ± 0.1 8.9 ± 0.4Blood glucose (mg %) 112.0 ± 7.0 123.1 ± 5.5Sodium (meq/l) 153.3 ± 0.5 149.5 ± 0.7*

Potassium (meq/l) 4.3 ± 0.6 5.0 ± 0.1WBC (·103 l/l) 6.9 ± 0.8 5.8 ± 0.7RBC (·106 l/l) 7.5 ± 0.1 7.6 ± 0.1Hemoglobin (g %) 13.9 ± 0.2 14.2 ± 0.1Hematocrit (%) 44.9 ± 0.8 47.6 ± 0.7*

MCV (fQ) 60.0 ± 0.8 62.5 ± 1.1Platelets (·103 l/l) 639.0 ± 45.3 831.0 ± 33.9*


treated orally with 100 mg/kg body weight drug dose for30 days are given in Table 7. In drug treated animals therewas no change in any organ weight/body weight ratios incomparison to control.

The adrenals, heart, kidneys, lung and spleen showed anormal histological appearance in rats of all the groupsexamined. The liver samples showed mild vacuolizationof the periportal hepatocytes in rats administered seabuck-thorn leaf aqueous extract at doses of 1 g/kg and 2 g/kgbody weight, single dose per day for 14 days (Fig. 4).

4. Discussions

Herbal medicines are used by up to 80% of the popula-tion in the developing countries. Despite the widespreaduse, few scientific studies have been undertaken to ascertainthe safety and efficacy of characterized and calibratedtraditional remedies. The seabuckthorn leaves aqueousextract used in this study was partially characterized withreference to HPLC fingerprinting and total phenolic con-tents. The extract was found to be rich in high phenoliccontent (363 mg/g of gallic acid equivalent) which may beresponsible for the observed adaptogenic activity. In thepresent dose dependent study of seabuckthorn leaves aque-ous extract a single oral dose of 100 mg/kg was found toprovide maximum adaptogenic activity in C–H–R animalmodel. There is only one study available on seabuckthornleaves related to its anti-stress and adaptogenic properties(Geetha, 2004). In this study 70% alcoholic extract of sea-buckthorn leaves, obtained by soxhlet extraction was eval-uated using C–H–R animal model and a single oral dose of100 mg/kg body weight, 30 min prior to exposure, pro-vided about 70% resistance to C–H–R induced hypother-mia by delayed fall of Trec 23 �C (112 ± 11.4 min vs65 min in control) and 33% faster recovery (123 ±4.6 min vs 184 ± 8.4 min in control) (Geetha, 2004). Thereported results of 70% alcoholic extract (Geetha, 2004)and present study observations of aqueous extractappeared to be comparable in providing resistance to C–H–R induced hypothermia but aqueous extract providedmuch faster recover of Trec 37 �C (44%) as observed inthe present study. Five doses administration, single doseper day, of the maximal effective dose (100 mg/kg body

Fig. 4. Light microscopic evaluation of liver tissues of control and experimental rats orally administered seabuckthorn aqueous leaf extract (H&E) ·400.(A) High power view of liver of control rat showing the portal triad structures and the periportal area. Cords of liver cells with sinusoidal spaces in betweenare seen. (B) High power view of liver of rat administered 1 g/kg body weight dose of seabuckthorn aqueous leaf extract, single dose/day for 14 days,showing vacuolated hepatocytes around the portal triad. (C) High power view of liver of rat administered 2 g/kg body weight dose of seabuckthornaqueous leaf extract, single dose/day for 14 days, showing vacuolated hepatocytes.


weight) provided adaptogenic activity similar to single doseresults. This suggested that the seabuckthorn leaves aque-ous extract do not possess cumulative adaptogenic activity.

The sub-acute 30 days administration of 100 mg/kg bodyweight dose, the maximal effective adaptogenic dose, wasobserved to be safe as body weight gain of the experimentalanimals was comparable with controls. The biochemicalparameters of experimental animals were also within thenormal range and comparable with controls showing noindication of drug induced damage. Though there was a sig-nificant decrease in serum sodium levels but the values werewith in the normal range. In hematological parametersstudied the observed increase in hematocrit value may beon account of observed non significant rise of mean corpus-cular volume (MCV). In the present study experimental ani-mal’s serum LDH and creatinine levels were observed to becomparable with controls, suggesting that seabuckthornleaf aqueous extract sub-acute administration had no effecton cell membrane permeability, muscle metabolism andkidney function. The comparable organ weight/bodyweight ratios of all the organs of experimental and controlanimals suggested that there was no gross toxic effect ofthe extract on any vital organs when the maximal effectivedose was administered for 30 days.

In sub-acute toxicity studies of 10 and 20 times ofmaximal effective dose (1 g and 2 g/kg body weight), admin-istered for 14 days, the body weight gain and biochemicalparameters related to toxicity viz. serum bilirubin, creati-nine, glutamic pyruvic transaminase (GPT) were unalteredand comparable to controls. It is well known that livermetabolizes a wide range of both exogenous and endoge-nous compounds and act as a good indicator of detoxifica-tion process in the organisms. The unaltered GPT levelssuggested that there was no liver toxicity in rats due toadministration of seabuckthorn leaves aqueous extract athigher doses of 1 g/kg and 2 g/kg body weight, single doseper day for 14 days. But the increased liver organ weight/body weight ratio in rats administered 1 g/kg body weightdose and decreased kidney organ weight/body weight ratiosuggested that aqueous seabuckthorn leaf extract adminis-tration at higher concentration and for long duration mightbe affecting hepatic and kidney functions. The kidney of theexperimental animals administered 1 g/kg body weight doseshowed normal histological appearance, suggesting nokidney toxicity. But mild vacuolization of the periportalhepatocytes was observed in the histopathology of liversamples of animals administered 1 g/kg and 2 g/kg bodyweight doses, single dose per day for 14 days. However,


there were no degenerative changes in the hepatocytes, livernecrosis or inflammation seen in any of the samples exam-ined. Since the biochemical tests for liver function viz.GPT, did not show any abnormalities in any of the groupsthe histological observation of mild vacuolization is notconsidered to be significant. The results indicated that theuse of the herbal extract preparation in the used doses didnot lead to any significant toxicity in any of the studiedorgan.

In the present study, there was no change in any hema-tological parameters of the 1 g/kg body weight drug dosetreated experimental animals. However, in rats adminis-tered 2 g/kg body weight drug dose there was a significantincrease in RBC numbers which reflected in increasedhematocrit levels. It appears that a lower dose of100.0 mg/kg given for 30 days and its 20 time dose (2 g/kg) given for 14 days had an effect on hematologicalparameters. Probably, if a 10 time dose (1 g/kg) was alsogiven for longer duration of more than 14 days, might haveresulted in hematological parameters changes.

Acute toxicity refers the ability of the substance to dosystemic damage as a result of a one-time exposure to rel-atively large amounts of a substance. The objective of acutetoxicity studies is to determine the median lethal dose(LD50) after a single dose administration. LD50 value isused to show that 50% of the animals exposed to a specificamount of a substance died as a result. In the present studythe oral LD50 of seabuckthorn leaf aqueous extract in ratswas observed to be >10 g/kg body weight. Even after singledose treatment with 5 and 10 g/kg body weight dose onlyone rat died out of 12 rats (8.3%) in >24 h after the drugadministration. This suggested low toxicity of seabuck-thorn leaf aqueous extract, as larger is the LD50 value thelower the toxicity.

In conclusion, the present study suggested that the max-imal effective adaptogenic dose of seabuckthorn dry leavesaqueous extract in rats, administered orally 30 min prior toC–H–R exposure, was 100 mg/kg body weight while itsLD50 value was >10 g/kg body weight. The observedchanges in hepatic and kidney organ weight/body weightratios and histology of animals administered doses 10and 20 times of effective dose suggested that use of highdoses of seabuckthorn dry leaves aqueous extract forextended durations requires caution.

Conflict of interest statement

There are none.

References

Beveridge, T., Li, T.S.C., Oomah, B.D., 1999. Seabuckthorn products:manufacture and composition. J. Agric. Food Chem. 47, 3480–3488.

Bhargava, K.P., Singh, N., 1981. Antistress activity of Ocimum sanctum

Lin. Ind. J. Med. Res. 73, 443–451.Bonsnes, R.W., Taussky, H.H., 1945. On the colorimetric determination

of creatinine by the Jaffe reaction. J. Biol. Chem. 158, 581–591.

Brekhman II, 1980. Man and Biologically Active Substances. PergamonPress, Oxford.

Cheng, J., Kondo, K., Suzuki, Y., Ikeda, Y., Meng, X., Umemura, K.,2003. Inhibitory effects of total flavones of Hippophae rhamnoides L.on thrombosis in mouse femoral artery and in vitro platelet aggrega-tion. Life Sci. 72, 2263–2271.

Divekar, H.M., Radhey, S., Gupta, A.K., Pahwa, M.L., Grover, S.K.,Kumar R., Srivastava, K.K., 1996. Screening of Adaptogenic Activityof some Laddakhi Plants. DIPAS (Min. of Defence, India) Report No.1/96.

Eccleston, C., Baoru, Y., Tahvonen, R., Kallio, H., Rimbach, G.H.,Minihane, A.M., 2002. Effect of an antioxidant rich juice (seabuckthorn) on risk factors for coronary heart disease in humans. J.Nutr. Biochem. 13, 346–354.

Fulder, S., 1980. The drug that builds Russians. New Sci. 88, 576–579.Ganju, L., Padwad, Y., Singh, R., Karan, D., Chanda, S., Chopra, M.K.,

Bhatnagar, P., Kashyap, R., Sawhney, R.C., 2005. Anti-inflammatoryactivity of seabuckthorn (Hippophae rhamnoides) leaves. Int. Immu-nopharmacol. 5, 1675–1684.

Geetha, S., Sai, R.M., Singh, V., Ilavazhagan, G., Sawhney, R.C., 2002.Anti-oxidant and immunomodulatory properties of seabuckthorn(Hippophae rhamnoides) – an in vitro study. J. Ethnopharmacol. 79,373–378.

Geetha, S., Sai, R.M., Mongia, S.S., Singh, V., Ilavazhagan, G., Sawhney,R.C., 2003. Evaluation of antioxidant activity of leaf extract ofSeabuckthorn (Hippophae rhamnoides L.) on chromium (VI) inducedoxidative stress in albino rats. J. Ethnopharmacol. 87, 247–251.

Geetha, S., 2004. Antioxidant, Immunomodulatory and AdaptogenicPotential of Sea buckthorn (Hipphophae rhamnoides L.) – in vitro &in vivo Studies. Ph.D. Thesis Submitted to Delhi University, Delhi, p.111.

Goel, H.C., Gupta, D., Gupta, S., Garg, A.P., Bala, M., 2005. Protectionof mitochondrial system of Hippophae rhamnoides L. against radiationinduced oxidative damage in mice. J. Pharm. Pharmacol. 57, 135–143.

Grover, S.K., Divekar, H.M., Kumar, R., Pahwa, M.L., Bhardwaj, S.K.,Gupta, A.K., Srivastava, K.K., 1995. Experimental evaluation of aComposite Indian Herbal Preparation II (CIHP II) as an adaptogenand its mechanism of action. Int. J. Pharmacogn. 33, 148–154.

Gupta, V., Gupta, A., Saggu, S., Divekar, H.M., Grover, S.K., Kumar,R., 2005. Antistress and adaptogenic activity of L-arginine supple-mentation. eCAM 2 (1), 93–97.

Kallio, H., Yang, B., Peippo, P., 2002. Effects of different origins andharvesting time on vitamin C, tocopherols and tocotrienols inseabuckthorn (Hippophae rhamnoides L.) berries. J. Agric. FoodChem. 50, 6136–6142.

King, J., 1965. Practical Clinical Enzymology. D. Van Nostrand, London,pp. 441–443.

Kornberg, A., 1969. Lactate dehydrogenase of muscle. Methods inEnzymology. Academic Press, New York, pp. 11–16.

Kumar, R., Divekar, A.K., Gupta, A.K., Shyam, R., Srivastava, K.K.,1996. Enhanced thermogenesis in rats by Panax ginseng, multivitaminsand minerals. Int. J. Biometeorol. 39, 187–191.

Kumar, R., Grover, S.K., Shyam, R., Divekar, H.M., Srivastava, K.K.,1999. Enhanced thermogenesis in rats by a composite Indian herbalpreparation-I and its mechanism of action. J. Altern. Complem. Med.5, 245–251.

Kumar, R., Shyam, R., Divekar, H.M., Pahwa, M.L., Srivastava, K.K.,2000. Mechanism of increased tolerance to hypothermia after com-posite Indian herbal preparation II administration. J. Altern. Comp-lem. Med. 6, 509–517.

Kumar, R., Divekar, H.M., Gupta, V., Srivastava, K.K., 2002. Antistressand adaptogenic activity of lecithin supplementation. J. Altern.Complem. Med. 8, 487–492.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Proteinmeasurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275.

Lowry, O.H., Roberts, N.R., Mei-Ling, W.U., Hixon, W.S., Crawford,E.J., 1954. The quantitative histochemistry of brain: quantitativeenzyme measurements. J. Biol. Chem. 207, 19–37.


Malloy, H.T., Evelyn, K.A., 1937. The determination of bilirubin with thephotoelectric colorimeter. J. Biol. Chem. 119, 481–490.

McManus, J.F.A., Mowry, R.W., 1965. Staining Methods: Histologicaland Histochemical. Harper and Row, New York.

Negi, P.S., Chauhan, A.S., Sadia, G.A., Rohinishree, Y.S., Ramteke,R.S., 2005. Antioxidant and antibacterial activities of various sea-buckthorn (Hippophae rhamnoides L.) seed extracts. Food Chem. 92,119–124.

Ramachandran, U., Divekar, H.M., Grover, S.K., Srivastava, K.K., 1990.New experimental model for evaluation of adaptogenic products.J. Ethnopharmacol. 29, 275–281.

Riley, V., 1960. Adaptation of orbital bleeding technique to rapid serialblood studies. Proc. Soc. Exp. Biol. Med. 104, 751–754.

Rousi, A., 1971. The genus Hippophae L., a taxonomic study. Ann. Bot.8, 177–227.

Singleton, V.L., Rossi, J.A., 1965. Colorimetric of total phenolic withphosho-molybidic-phospho-tungstic acid reagents. Am. J. Enol. Viti-cult. 16, 144–158.

Suleyman, H., Demirezer, L.O., Buyukokuroglu, M.E., Akcay, M.F.,Gepdiremen, A., Banoglu, Z.N., Gocer, F., 2001. Antiulcerogeniceffect of Hippophae rhamnoides L.. Phytother. Res. 15, 625–627.

Wroblewski, F., LaDue, J.S., 1956. Serum glutamic pyruvate transami-nase in cardiac and hepatic disease. Proc. Soc. Exp. Biol. Med. 91,569–571.

Xing, J., Yang, B., Dong, Y., Wang, B., Wang, J., Kallio, H.P., 2002.Effects of sea buckthorn (Hippophae rhamnoides L.) seed and pulp oilson experimental models of gastric ulcer in rats. Fitoterapia 73, 644–650.

Yang, B., Kalimo, K.O., Tahvonen, R.I., Mattila, L.M., Katajisto, J.K.,Kallio, H.P., 2000. Effect of dietary supplementation with seabuckthorn (Hippophae rhamnoides) seed and pulp oils on the fattyacid composition of skin glycerophospholipids of patients with atopicdermatitis. J. Nutr. Biochem. 11, 338–340.

Zeb, A., 2004. Chemical and nutritional constituents of sea buckthornjuice. Pak. J. Nutr. 3, 99–106.

1-s2.0-s0278691506002985-main

Documents