Safety profile of 40% Garcinol from Garcinia indica in experimental rodents

Highlights • Evaluated standardized 40% Garcinol in Wistar rats for its safety.• Acute, sub-acute sub-chronic and reproductive/developmental toxicity study conducted.• 40% Garcinol safe up to 2000 mg/kg at single dose.• NOAEL of 40% Garcinol at sub acute, sub-chronic and reproductive/developmental study is 100 mg/kg/day.


Introduction
Food and health are the two terms always associated with each other through ages and today life sciences contribute to explain it as characterized by complexity [1]. Also, benefit of food beyond the basic nutritional value in prevention of disease and health enhancement using its elements is the interest of people today. Most of the bioactive components related to food products used daily, originated in plant are owed by the traditional medicinal practice [2]. There is continuous recognition of unique structured low molecular weight primary compounds from the natural components when compared to the combinatorial chemistry. Only the 10% of biodiversity has been analysed for their biological activity and numerous are there to be evaluated [3].
Likewise, Garcinia indica from Clusiaceae [4] family commonly known as Kokum is an evergreen plant native to Western Ghats of India but distributed throughout the tropical regions of India, Africa and China [5]. Kokum rind has been used as the alternative for tamarind or lemon in the culinary use and its extract in the beverages, gourmet spice and as carminative [6]. Ayurvedic system of medicine recommends its use in scurvy, rheumatism, oedema, sore, heat strokes, dermatological conditions, contagious diseases and as emollient and demulcent [7].
Sami labs limited has standardized 40% Garcinol preparation for its efficacy studies at the doses not exceeding 10 mg/kg with significant outcomes (data not shown). It has various health benefits; progression of Garcinol into a pharmacologically active molecule needs further toxicological and pharmacokinetic studies which are required to establish the safety [20].
The present work was taken up to summarise acute, sub-acute, subchronic and reproductive/developmental toxicity studies of 40% Garcinol in the experimental animals.

Chemicals
The AR (Analytical Grade) solvents and chemicals were used in all toxicity studies. The 40% Garcinol i.e. test sample with batch no. C120775 was prepared by phytochemical group at Sami Labs Limited. The reference sample (C120775) has been stored at Sami Labs Limited.
Garcinol (Crystallized yellow needles) chemically polyisoprenylated benzophenone derivative of molecular formula C 38 H 50 O 6 ( Fig. 1) with molecular weight of 602.39252 and melting point of 122°C is obtained from Garcinia indica fruit rind [5,20]. Dried Garcinia indica fruit rind was extracted with hexane and fractionated by column chromatography. Hexane extract was completely crystallized in ethanol and vacuum dried. Thus, obtained residue of Garcinol was dissolved in cold hexane and re-crystallized at room temperature to obtain as pale yellow needle like crystals [10]. The acquired Garcinol from the above process was standardized to 40% with microcrystalline cellulose powder. The HPLC graph of standardized sample at 240 nm is as shown (Fig. 2).

Animals
Wistar Rats of 6-10 weeks old, housed maximum of 5 per sex per cage in a polypropylene cage with maintained room temperature of 20.2-23.5°C, relative humidity of 30-70% with 12 h fluorescent light and 12 h dark cycle were used in all the toxicity studies. Animals were fed with Nutrilab rodent feed (Provimi Animal nutrition Pvt. Ltd) and purified water (aqua guard water filter) that was provided ad libitum. Animals were kept in sterilized rice husk beddings changed along with the cage twice a week during acclimatization and entire experimental study period.

Ethics
Each separate protocol for each (acute, sub acute, sub chronic and reproductive/developmental toxicity) study was approved by Institutional Animal Ethics Committee of CSIR-Indian Institute of Toxicology Research, Lucknow (registration no. 54/99/CPCSEA) and of Bioneeds, Bangalore (registration no. 969/bc/06/CPCSEA) respectively. The studies were conducted in accordance with the recommendation of the committee for the purpose of control and supervision of experiments on animals (CPCSEA) guidelines for laboratory animal facility published in the gazette of India, December 15th 1998. Also the studies were in compliance with OECD principles of good laboratory practice, guidelines for testing of chemicals no.420, 407, 408 and 421 and in accordance with SOP of the institution.

Acute oral toxicity study
Required quantity of 40% Garcinol was weighed and then mixed with 0.5% w/v Carboxy methyl cellulose. Volume was made up with 0.5% w/v Carboxy methyl cellulose in the measuring cylinder in accordance with the required dose. The 40% Garcinol was administered orally by gavage as single dose using gavage needle number 14. Dose was adjusted according to the body weight which was measured on the dosing day and dose volume was 10 ml/kg. Food was offered 3-4 hrs after dosing. The dose given was 300 mg/kg body weight, 2000 mg/kg body weight and 2000 mg/kg body weight in sighting study step-I, sighting study step-II and main study respectively. All the animals were observed for clinical signs of toxicity and mortality at 30-40 min, 1 h ( ± 10 min), 2 h ( ± 10 min), 3 h ( ± 10 min) and 4 h ( ± 10 min) on the day one after dosing and thereafter once daily for clinical signs and twice daily for mortality during the 14 days study period. All the test animals were weighed on day 1 before the dose administration and on day 7 and 14 during the study period. All the animals were sacrificed at term by overdose of CO2 and subjected to necroscopy.

Sub acute or 28 days repeated dose toxicity study
The formulation of 40% Garcinol was prepared fresh daily and vehicle was administered at the dose volume of 10 mg/kg body weight. The animals were grouped 20, 50 and 100 mg/kg/day (Table 1) as there was no statistically significant toxicity in single dose toxicity and not more than 5-10 mg/kg dose showed effect in the efficacy study (data not shown). The dose volume was calculated for individual animals according to body weight during the treatment period. Dose administration was done by oral gavage once daily for four weeks. There was no administration of 40% Garcinol formulation/vehicle during the 14 days recovery period.   Rats were observed for clinical changes and were recorded prior to the dose formulation and weekly during the study period. Changes in skin and fur, eyes, mucous membrane, occurrence of secretions and excretions, changes in gait, posture and presence of any change in general behaviour of animals were observed. Individual body weights were recorded before dosing (day 1) and weekly thereafter. Fasting body weights were recorded before sacrifice. Body weights were recorded from recovery animals after cessation of dosing, during the recovery period. Fasted body weights were used for calculation of organ: body weight ratio. The food consumption was measured as average cage-wise food intake (g/rat/day) at weekly intervals during in-life phase of the experiment.
The collection of blood sample at the end of study period (28 th day for main groups & 42 nd day for recovery groups) was done into K 2 EDTA tubes for haematology and tubes without anticoagulant for clinical chemistry. Haematological parameters were determined using Sysmex XT 1800 IV haematology analyzer and biochemistry parameters analyzed using Rx Daytona (Randox) automatic analyzer after serum was separated in a refrigerated centrifuge at approximately 5000 rpm for 10 min. The finding from detailed necropsy of all group rats was recorded and the animals were examined visually for external abnormalities including palpable masses. Tissues and organs collected on completion of the gross pathology examination from all animals were preserved in 10% formaldehyde.

Sub chronic or 90 days repeated dose toxicity
The dose grouping of the animals is as shown in Table 2. The particular doses were taken with reference to the 28 days toxicity study as there was no dose related toxicity so to observe its effect in long term. The individual animal dose was determined by the individual weight taken before dosing and weekly thereafter during the study period. The 40% Garcinol or vehicle formulation was administered by oral gavage to all the groups once daily at approximately the same time each day for a period of thirteen weeks. There was no administration of 40% Garcinol formulation/vehicle during the 14 days recovery period.
Rats were observed for clinical changes and were recorded prior to the dosing and weekly during the study period. Changes in skin and fur, eyes, mucous membrane, occurrence of secretions and excretions, changes in gait, posture and presence of any change in general behaviour of animals were observed. Fasting body weights were recorded before sacrifice. Body weights were recorded from recovery animals after cessation of dosing, during the recovery period. Fasted body weights were used for calculation of organ: body weight ratio. The food consumption was measured as average cage-wise food intake (g/rat/ day) at weekly intervals during in-life phase of the experiment.
The collection of blood sample at the end of study period (90 days for main groups and 104 days for recovery groups) was done into K 2 EDTA tubes for haematology and tubes without anticoagulant for clinical chemistry. Haematological parameters were determined using Sysmex XT 1800 IV haematology analyzer and biochemistry parameters analyzed using Rx Daytona (Randox) automatic analyzer after serum was separated in a refrigerated centrifuge at approximately 5000 rpm for 10 min. The finding from detailed necropsy of all group rats was recorded and the animals were examined visually for external abnormalities including palpable masses. Tissues and organs collected on completion of the gross pathology examination from all animals were preserved in 10% formaldehyde.

Reproduction/developmental toxicity study
The vehicle used to make up the volume of 40% Garcinol was Carboxy methyl cellulose and dose grouping was done as shown in Table 3. The dose formulations and vehicle was administered according to body weight everyday for a period of 14 days prior to mating, during mating and in females during mating, pregnancy and day 4 of post partum of pups. One male and one female rat were mated (1:1), the female was placed with same male until pregnancy occurs or two weeks were elapsed. Each morning females were examined for the presence of sperm or vaginal plugs. Day 0 of pregnancy is considered as the day a vaginal plug or sperm were found. In case pairing is unsuccessful, remating of females with proven males of same group was considered.
Rats were observed for changes as in sub chronic study for the presence of abnormal behaviour, if any. Body weights of the animals were taken on day 1 of dosing and weekly thereafter. During gestation period, females were weighed on days 0, 7, 14 and 20, after parturition and day 4 postpartum. Pups at birth and day four were counted and weighed individually. All the surviving animals were sacrificed and were examined microscopically. The animals were examined visually for external abnormalities and microscopic examination of reproductive organs. The counting of implantation sites and number of corpora lutea was done. The tissues were processed for routine paraffin embedding and sections were stained with haematoxyline and Eosin statin. Histopathological examination of low dose and mid dose was not carried out as preserved organs of vehicle control and high dose group rats didn't show gross histopathological changes.

Statistical analysis
The data generated was subjected to statistical evaluation for the significance of any 40% Garcinol induced changes and their interpretation of potential for toxicity. The statistical analysis of the experimental data was carried out by the one-way ANOVA test. Statistical comparisons were evaluated at the 5% (P ≤ 0.05) significance level. For reproductive/developmental toxicity studies, all quantitative variables like laboratory investigations (pups size and pups survival ratio) were subjected to Fishers Student T-test and Wilcoxon Rank Sum test. In the case of recovery groups also, data was analyzed using the methods stated above. Comparison of means between treatment groups was  performed.

Results
In acute oral toxicity study with the single high dose of 2000 mg/kg body weight didn't show any significant clinical signs and mortality in the female Wistar rats. The body weight gain was comparable during study period as shown in Table 4.
In 28 days repeated dose toxicity study, there were no clinical signs of toxicity and mortality noticed in any of the treatment or control group animals. There were no differences in the body weight (Fig. 3), body temperature and food consumption changes between test and control group during the course of study and recovery period. The haematological parameters (Table 5), biochemical parameters (Table 6), pathological examination and organ weight ( Table 7) at necropsy showed no significant changes.
In 90 days repeated dose toxicity study, there were no abnormal clinical signs and mortality in any of the treatment or control group animals. There was no significant change in body weight gain over the study period (Fig. 4). The haematological parameters (Table 8), biochemical parameters (Table 9), absolute and relative organ weight (Table 10) of the animals are as shown.
In reproductive and developmental toxicity study, none of the animals in any groups, both control and test item treated exhibited any abnormal clinical signs/behavioural changes and mortality that may be attributed to the test item. There was no loss in body weight or difference in body weight (Tables 11 and 12) as well as organ weight (Table 13) between control and treated groups during the study period. The reproductive/developmental parameters of the animals were comparable as shown in the Table 14.

Discussion
Garcinol, a compound found in the Garcinia species fruit rind is derivative of polyisoprenylated benzophenone class [5]. It has NAD-no abnormality observed.  antioxidant, anticancer, anti HIV; antiulcer activities studied so far [3].
Although there are many preclinical efficacy studies being conducted on Garcinol [3,[9][10][11][12][13][14][15], there are no published toxicity profiles of this molecule. We are in process of evaluating Garcinol 40% for its efficacy and thereby establishing its safety becomes of paramount importance. So we have taken up this work to study pre-clinical safety profile of standardized 40% Garcinol.
In acute single dose toxicity study, female Wistar rats were observed for clinical signs of toxicity and mortality, feed consumption, body weight gain and gross pathology and found to be normal during the study period. Further observation of animals for 14 days revealed no toxic effects. Thus, it is considered 40% Garcinol to be non-toxic at the single high dose of 2000 mg/kg.
Similarly, no abnormal clinical signs and mortality was observed in the 28 days sub acute study. Body weight gain, body temperature and feed consumption didn't have differences in the control and 40% Garcinol treated group during the study period and in the recovery groups too. There were biologically insignificant changes in some of the Values are in mean, M-Male, F-Female, n = 5. haematological parameters in the few animals which were comparable to the respective controls. There was biologically insignificant decrease in creatinine and urea of female animals of mid dose groups. In the histopathological examination, few animals presented minor lesions which didn't reveal dose response with respect to severity which shows that 40% Garcinol is non-toxic during 28 days sub acute toxicity study.
As there is potential long term use of the molecule, we established the long term safety of 40% Garcinol by taking up the 90 days sub chronic toxicity study. All the animals were observed for clinical signs, changes in body weight and feed consumption daily and were  comparable among the control and treatment groups. In female animals a decrease was noticed in the WBC and MCHC levels in the high dose recovery groups and remaining parameters were unaffected. The increase in the clinical parameters was not distributed in dose related pattern and was in between physiological limits. The absolute and relative organ weight of the animals were comparable among the treated and control group. In histopathological examination, there were few spontaneous and incidental lesions which were comparable with Table 9 Effect of 90 days exposure to 40% Garcinol on biochemical parameters in Wistar Rats.
Values are mean ± SD, M-Male, F-Female, n = 10, P values: * < 0.05 (values of low, mid and high dose group were compared with control). Values are mean ± SD, M-Male, F-Female, n = 10, P values: * < 0.05 (values of low, mid and high dose group were compared with control). In reproductive and developmental toxicity study, there were no mortalities and clinical signs of abnormalities during the study period in any of the groups. The mean body weight of females in the gestation as well as during 4 days lactation period was comparable to control group females (Fig. 5). Further, there were no 40% Garcinol related histopathological changes in reproductive organs (testes, epididymus, ovary, uterus, seminal vesicles) of the high dose animals as well the organ weights were comparable to the control group animals. The reproductive parameters like females showing evidence of copulation, females achieving pregnancy, time taken to conceive were normal indicating lack of reproductive toxicity of 40% Garcinol. Pre-implantation and post implantation, loss at the birth and up to day 4 post partum were comparable in all groups. One female each from all the control and dose groups were non pregnant which didn't affect the outcome of the study as per the guideline (minimum 8 pregnant animals per group). The number of live pups on the day of birth and day 4 were not changed as there was no loss of pups post partum. The male to female ratio and body weight of pups were comparable among the 40% Garcinol treated and control groups. Thus, the result obtained shows that use of standardized 40% Garcinol in the reproductive/developmental stage of the Wistar rats is safe with no observed adverse effect.

Conclusion
The 40% Garcinol did not show any adverse effect at high single dose of 2000 mg/kg at acute safety study in Wistar rats and can be classified as GHS category 5/unclassified according to the Globally Harmonized classification system (GHS) for classification of chemicals. There were no treatment related changes induced by 40% Garcinol, at highest dose of 100 mg/kg/day at 28 days repeated dose oral toxicity study, 90 days repeated dose oral toxicity and reproductive/developmental toxicity study. Thus, it can be concluded that 40% Garcinol has low toxicity profile in rodents and had no observed effects under experimental conditions applied.

Transparency document
The Transparency document associated with this article can be Values are Mean ± SD of 9 animals. Values are Mean ± SD, n = 10. found in the online version.