SAFETY ASSESSMENT OF PARTIALLY PURIFIED HDL ENHANCING PHYTOCOMPONENTS FROM DESMODIUM GYRANS DC.

Desmodium gyrans (Fabaceae) is a less explored plant belonging to medicinally important genera Desmodium. A recent investigation has reported hypolipidemic and HDL enhancing efficacy of crude alcoholic extract (DGM) of the whole plant. Chromatographically purified aqueous fraction (DGMAF) rich in terpenoids and phenolics exhibited HDL enhancing and reverse cholesterol transport (RCT) promoting activity that may impart atheroprotective potential to this active fraction. Present study was aimed to evaluate the acute and sub-acute oral toxicity of DGMAF in Wistar rats of either sex following OECD guide lines to ensure its safety upon long term exposure. Acute oral dose of 5g/kg body weight did not produce any behavioural abnormality, weight changes or mortality over 14 day period. Necropsy studies revealed no incongruity to internal organs. Further, oral sub-acute doses of 100, 250, and 500 mg/kg body wt for 60 days did not show reduction in body and organ weight, food and water intake. Hematological parameters and biochemical analytes inclusive of hemoglobin, urea, creatinine, total and direct bilirubin, serum GOT, GPT, and ALP did not show statistically significant differences between the untreated control and treated groups. No morphological changes were observed in the histological analysis of the major vital organs including liver, kidney, spleen and intestine. The result of this study thus suggests that DGMAF might be non-toxic at normal therapeutic doses. Activities of serum marker enzymes such as total and direct bilirubin, glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP) were estimated using commercially available kits obtained from Span Diagnostics Ltd (Surat, India). Kidney function tests such as serum creatinine and urea were also measured using kits from Span Diagnostics.

Desmodium gyrans (Fabaceae) is a less explored plant belonging to medicinally important genera Desmodium. A recent investigation has reported hypolipidemic and HDL enhancing efficacy of crude alcoholic extract (DGM) of the whole plant. Chromatographically purified aqueous fraction (DGMAF) rich in terpenoids and phenolics exhibited HDL enhancing and reverse cholesterol transport (RCT) promoting activity that may impart atheroprotective potential to this active fraction. Present study was aimed to evaluate the acute and sub-acute oral toxicity of DGMAF in Wistar rats of either sex following OECD guide lines to ensure its safety upon long term exposure. Acute oral dose of 5g/kg body weight did not produce any behavioural abnormality, weight changes or mortality over 14 day period. Necropsy studies revealed no incongruity to internal organs. Further, oral subacute doses of 100, 250, and 500 mg/kg body wt for 60 days did not show reduction in body and organ weight, food and water intake. Hematological parameters and biochemical analytes inclusive of hemoglobin, urea, creatinine, total and direct bilirubin, serum GOT, GPT, and ALP did not show statistically significant differences between the untreated control and treated groups. No morphological changes were observed in the histological analysis of the major vital organs including liver, kidney, spleen and intestine. The result of this study thus suggests that DGMAF might be non-toxic at normal therapeutic doses.

Introduction:-
Herbal products are attaining popularity as alternative medicine for treatment of various diseases because of its effectiveness, low cost and safety on long term use. The methanolic extract of D. gyrans (DGM) has been previously shown to reduce high fat diet induced hyperlipidemia in rats and rabbits with significant increase in HDL fraction (Vipin et al., 2015). Further to confirm the RCT promoting efficacy DGMAF has to be tested in-vivo using rat models. In previous reports, there is no record in the literature on the toxicity profile of Desmodium gyrans. The acute and sub-acute toxicity data are mandatory to predict the safety and effects of long term exposure. The present study was therefore undertaken to determine the toxicity profile of DGMAF on Wistar rats. This study evaluates DGMAF for its toxic effects using hematology, serum parameters, and histopathological changes as toxicity indices.

Acute toxicity analysis of DGMAF:-
The oral acute toxicity of DGMAF was evaluated according to Organization for Economic Co-operation and Development (OECD) guideline. All rats were kept at room temperature with persistent humidity. They were permitted to acclimatize to laboratory conditions for a week prior to the experiment. Drinking water and food were provided ad libitum throughout the experiment, except for the short fasting period where the drinking water was still in free access but no food supply was provided 12 hr prior to treatment. Animals were observed for 7 days before oral drug administration. Following the fasting period, body weights of the rats were determined and the dose was calculated in reference to the body weight. After 7 days, a single high dose of 5 g/kg. b.wt of DGMAF (dissolved in distilled water) was administered to three female and three male rats (Average weight: 189 g) by the oral route. Foods were provided to the rats approximately an hour after treatment. The rats were observed in detail for any indications of toxicity effect within the first six hours after the treatment period, and daily further for a period of 14 days. Surviving animals were weighed and visual observations for mortality, behavioral pattern, changes in physical appearance, injury, pain and signs of illness were conducted daily during the period. At the end of experimental period the animals were euthanized and necropsy was carried out.
Sub-acute toxicity:-Wistar rats of both sexes (males and females) were used for the study. During the experimental period of 60 days, the rats were grouped into four groups comprising untreated normal and drug groups such as doses of 100 mg/kg b.wt, 250 mg/kg b.wt and 500 mg/kg b.wt of DGMAF. During the experimental period, food and water consumption and increase in body weight was recorded once weekly. At the end of the experimental period, the animals were fasted overnight and sacrificed on the following day. Blood was collected through cardiac puncture and used for the assessment of hematological parameters and organs including liver, kidney, lung, spleen, heart and brain were excised to calculate relative organ weight as well as histological analysis. Serum was obtained after centrifugation of blood at 1500 rpm for 10 min and used for biochemical analysis.
Hematological parameters such as total count and differential leukocyte count were assessed using whole blood. Hemoglobin was determined using Drabkin's method (Drabkin DL, 1932). Activities of serum marker enzymes such as total and direct bilirubin, glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP) were estimated using commercially available kits obtained from Span Diagnostics Ltd (Surat, India). Kidney function tests such as serum creatinine and urea were also measured using kits from Span Diagnostics.

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Histological analysis:-A portion of liver, kidney, spleen and intestine from each group were cut, washed in PBS, fixed in10% neutral buffered formaldehyde solution and then embedded in paraffin wax. Sections round 5-6 microns in thickness were taken and stained using double staining method using hematoxylin (nuclear stain) and eosin (cytoplasmic stain). Slides were then photographed at 400 X magnification.
Statistical analysis:-Data are presented as mean ± SD of 6 animals. Statistical comparisons are made using a one-way analysis of variance (ANOVA) followed by Dunnett't' test using Graph Pad Instat 3 software (Graph Pad Software, Inc. La Jolla, USA).

Results:-
Acute-toxicity study:-Single dose of DGMAF (5 g/kg b.wt) was given for acute toxicity based on OECD guideline 420 (Adopted December 2001), Para 19. There was no significant difference between the test groups administered with DGMAF in relation to the normal untreated group. In the acute toxicity analysis, the DGMAF was found to be non-toxic to Wistar rats of both sexes as no mortality or abnormal changes such as changes in skin and fur, eyes and mucus membranes, behavior pattern, tremors, salivation, sleep, coma, moribund, ill health or any visible reaction to treatment were observed during the period of two weeks. The body weight was maintained without much variation ( Figure 1) and no diarrhea or hair loss was observed. Also no significant changes were seen in hematological data such as hemoglobin concentration and total count of leukocytes (Table 1). Since there was no toxicity observed, doses above 5gm/kg b.wt is not considered for further assessment and has been assumed as its LD 50.  Sub-acute toxicity:-On oral administration of DGMAF for 60 days at doses of 100, 250 and 500 mg/kg b.wt, no mortality was seen in any of the groups. The animals were found to be generally healthy with no signs of toxicity during the observed 213 period of study. No abnormal changes in body weight ( Figure 2) or relative organ weight (Table 2) and food consumption ( Figure 3) were seen upon comparing with that of normal rats.   Hematological parameters such as Hb level, total WBC count and differential counts were found to be within normal limits in all groups (Table 3). Similarly, biochemical parameters remained unaffected as the level of liver function tests (Table 4), kidney function tests (Table 5) were also within normal values having no abnormalities of statistical significance. Necroscopy of animals after 60 days did not produce any anatomical changes in rats.   Histological examination of organs such as spleen, liver, intestine and kidney of all the DGMAF treated groups did not show any differences when compared with untreated groups indicating that no adverse toxicological effects had occurred in these organs ( Figure 4). In liver the hepatic architecture and arrangement of hepatocytes were not altered by DGMAF treatment and no change was observed compared to the normal group (Figure 4a). Appearance of glomerular tufts and tubules of kidney showed no differences between the treated and normal groups (Figure 4b).
Spleen and intestinal villi also did not show any change in appearance (Figure 4c and 4d). No necrosis was observed in the histological study during the treatment period.

Discussion:-
Natural products therapies are supposed to be harmless and less injurious to the human body than the synthetic drugs (Alam et al., 2011). Some pharmacological and toxicological data exist for these types of natural medicines (Fragoso et al., 2008). However, their safety has repeatedly been probed due to reported complaints and fatality on the test animals (Park et al., 2010). So it is significant to determine the safety of these remedies in order to support their use. Acute toxicity is an initial screening step, mandatory in the toxic assessment and evaluation of all biological compounds (Akhila et al., 2007) and it establishes the moderate lethal dose (LD 50 ) of biological compounds (Robinson et al., 2007). Acute toxicity is usually defined as the variations occurring instantly or a short time following a single administration of compound within 24 hrs (OECD, 2000).
In the present acute toxicity study, Wistar rats of both sexes were treated with DGMAF up to a dose of 5g/kg b.wt and no mortality or any signs of toxicity or side effects were recorded. In acute toxicity testing, doses higher than 5g/kg b.wt are generally not considered as dose related toxicity (Hayes, 1987). According to the OECD guidelines (Guidance Document for Acute Oral Toxicity Testing; OECD, 2001), compounds with LD 50 values above 2 g/kg b.wt are generally considered to be relatively safe. Thus, DGMAF can be considered to be non-toxic at acute administration as the fractions are well tolerated and there has been no observed harmful effect.

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In sub-acute toxicity study, following administration of DGMAF at 100, 250 and 500 mg/kg b.wt for 60 days, the body and organ weight are found to be normal without harming liver, blood or kidney tissue physiology or its tissue architecture. Change in organ-to-body weight ratio is the indication of organ damage (Busari et al., 2015). Comparison of organ weights between treated and control group of animals have conventionally been used to assess the toxic or harmful effects of drugs (Boyd, 1966 ;Pfeiffer, 1968). DGMAF does not have any harmful effects on rats that would cause them to loose appetite, thereby causing a reduction in food intake and subsequently a reduction in weight with increase in dose. Assessment of liver and kidney function is very important in evaluating the toxicity of plant extracts and an elevation in liver enzymes (ALT, AST and ALP) activity is conventionally an indicator of liver injury (Chavda et al., 2010). In the present study liver marker enzymes, blood urea and creatinine levels are found to be unaltered in DGMAF treated animals over the 60 days period.
Hematological changes such as anemia are often accompaniments of bone marrow toxicity and analysis of blood parameters with respect to animal studies have a high relevance and predictive value for humans (Hamid Rhiouani et al., 2008). In this study hemoglobin and total differential count in the blood remained the more or less the same indicating the non-toxicity of DGMAF.

Conclusion:-
In conclusion, the present investigation demonstrates that at doses consumed in the traditional medicine, the aqueous fraction of methanolic extract of D. gyrans may be considered as relatively safe, as it does not cause either mortality or produce severe toxicological effects on selected body organs, biochemical indices and hematological markers of rats during the acute and sub-acute periods of study. These findings are very important as DGMAF with its hypolipidemic properties have the potential to be considered as a safer drug candidate for long term use in the treatment of atherosclerosis.