Evaluation of impact of Siddha Suthi (purification) processes on nut of Serankottai ( Semecarpus anacardium L.)

impurities unwanted toxic Abstract Introduction: Serankottai ( Semecarpus anacardium L.) is a Schedule E (1) drug and is considered for treating all kinds of Vatha diseases, venereal disease, skin disease and cancerous conditions. Suthi murai denotes the purification process before any drug is employed in medicine. This study is aimed at evaluating the impact of Siddha purification processes on macro-microscopical and physico-chemical characteristics of Serankottai so that a justification for such classical processes can be derived. Materials and Method: The raw (S1) and purified Serankottai (S2 to S6) samples were analyzed for their macroscopic, microscopic and powder microscopic analysis followed by the physicochemical parameters like loss on drying (LOD), total ash (TA), water soluble ash (WSA), acid insoluble ash (AIA), water soluble extractive (WSE), alcohol soluble extractive (ASE) and pH values. Results: Prismatic crystals, oil globules, sclereids, resin, fiber were observed in the microscopical studies of raw sample and these are found in the purified samples except S2 which has revealed charred cell structure. Physical nature of Serankottai has been maintained in all the purification methods except for S2. The LOD was 5.53%, 2.90%, 6.47%, 5.03%, 4.6%, 5.05% in sampleS1, S2, S3, S4, S5 and S6 respectively. The TA values were 3.44%, 18.90%, 2.59%, 2.54% 2.7% and 3.65% in sample S1, S2, S3, S4, S5 and S6 respectively. Significant differences in physico-chemical parameters were observed in different purification methods. Conclusion: The present study revealed that the Siddha purification processes have impact on physicochemical characters of Semecarpus anacardium . The need of the purification, as mentioned in Siddha texts is hereby justified.


Introduction
Serankottai or oriental cashew nut (dried nut with peduncle of Semecarpus anacardium L. -Anacardiaceae) is also called as marking nut since the black colored pigment from the nuts is used to make marks in fabrics for identification by the washer men (1).It is a moderate-sized deciduous tree found in the outer Himalayas and hotter parts of India up to 3500 ft (2).The important constituents of S. anacardium are bhilawanols, phenolic compounds (3,4), bioflavonoid(5), anacardoside (6), and semecarpetin (7). S. anacardium has been proved to possess important pharmacological activities like anti-inflammatory (8)(9)(10)(11)(12), antiartherosclerotic (13), antioxidant (14,15), CNS n o o t r o p i c ( 1 6 ) , h y p o g l y c e m i c ( 1 7 ) , a n d anticancer (18,19).
The Drugs and Cosmetics Act, 1940 categorized some of the Siddha plant drugs under Schedule E (1) classifications, so that these drugs are toxic and advised to take with more precautions. It is recommended to assure these drugs undergo purification process before they are used in medicinal preparation. Serankottai, a Siddha drug is one among the Schedule E (1) drugs (20).
Siddha system emphasizes the Suthi (purification) process of a drug before it is incorporated in any medicinal preparations. The purification process is to remove toxic content before it is used in medicine to counteract the toxic symptoms and to enhance its efficacy is termed as Suthimurai (purification process) in Siddha system of medicine. Purification processes are employed for all poisonous drugs from herbs, minerals, metals and animal origins and it is r e c o m m e n d e d f o r a l l d r u g s t o r e m o v e their kutram(impurities or unwanted toxic content). The purification is done by various pharmaceutical procedures like boiling, frying, washing, triturating with various plant juices; pudam (calcination) method;

Abstract
Introduction: Serankottai (Semecarpus anacardium L.) is a Schedule E (1) drug and is considered for treating all kinds of Vatha diseases, venereal disease, skin disease and cancerous conditions. Suthi murai denotes the purification process before any drug is employed in medicine. This study is aimed at evaluating the impact of Siddha purification processes on macro-microscopical and physico-chemical characteristics of Serankottai so that a justification for such classical processes can be derived. Materials and Method: The raw (S1) and purified Serankottai (S2 to S6) samples were analyzed for their macroscopic, microscopic and powder microscopic analysis followed by the physicochemical parameters like loss on drying (LOD), total ash (TA), water soluble ash (WSA), acid insoluble ash (AIA), water soluble extractive (WSE), alcohol soluble extractive (ASE) and pH values. Results: Prismatic crystals, oil globules, sclereids, resin, fiber were observed in the microscopical studies of raw sample and these are found in the purified samples except S2 which has revealed charred cell structure. Physical nature of Serankottai has been maintained in all the purification methods except for S2. The LOD was 5.53%, 2.90%, 6.47%, 5.03%, 4.6%, 5.05% in sampleS1, S2, S3, S4, S5 and S6 respectively. The TA values were 3.44%, 18.90%, 2.59%, 2.54% 2.7% and 3.65% in sample S1, S2, S3, S4, S5 and S6 respectively. Significant differences in physico-chemical parameters were observed in different purification methods. Conclusion: The present study revealed that the Siddha purification processes have impact on physicochemical characters of Semecarpus anacardium. The need of the purification, as mentioned in Siddha texts is hereby justified. grinding with specific organic or inorganic materials; soaking in specific media etc to minimize the toxicity of the substances and to strengthen the efficacy of the drug.
Purification is done to reduce the toxicity of the drug by potentiating the conversion of high toxic components to less toxic molecules or chemical structure.Siddha classical text have emphasized various Suthi muraigal (purification methods) to overcome the unwanted effects from various poisonous and nonpoisonous drugs, involving different media specific to s u b s t a n c e s s u c h a s p a s u m c h a a n a m (cow dung), komium (cow's urine), erumai chaanam (buffalo dung), arisi kaadi (rice vinegar), herbal kasayams (decoctions), herbal juices like Aloe vera juice, fruit juices like lemon juice,etc.
In the present study, different forms of S. anacardium nuts (unpurified and purified nuts following different Siddha methods of purification - Table 1) were analyzed to evaluate the macromicroscopic and physicochemical characters.

Materials and methods
The nuts of S. anacardium with peduncles were purchased from raw drug store K. Ramasamy Chetty country raw drug store, Paris corner, Chennai and authenticated at the Department of Pharmacognosy, SCRI, Chennai. The raw sample of Serankottai (S1) was analyzed followed by samples purified by five processes (S2, S3, S4, S5, and S6) mentioned in classical Siddha texts ( Table 1). The samples were analyzed for macro-microscopical and physiochemical parameters and the results were discussed.

Macro-microscopic examinations
The nuts of Semecarpus anacardium of six samples were subjected to macroscopic, microscopic and powder microscopic characters following standard procedures (26,27).

Physico-chemical Analysis
Physicochemical studies of the unpurified SA nut sample S1and purified SA nut samples S2 to S6 were done according to the PLIM guidelines (26,27).

Organoleptic characters
The organoleptic characters of the sample drug were evaluated in self. The samples S1-S6 were taken and the color, texture, smells, taste were tested (26,27).

Loss on Drying
Accurately weighed 4 g of the powdered drug was dried in the oven at 105°C to constant weight and percentage moisture content was calculated.

Determination of Ash Values Total Ash
Four grams of the test drug was accurately weighed and incinerated in silica crucible at 600 o C until free from carbon. It was then cooled and weighed. The % w/w of ash with reference to the air dried powder was calculated.

Water Soluble Ash
The total ash was obtained as per the above mentioned method. The ash was boiled for 5min with Process Method with classical reference Sample 1 The peduncles removed SA nuts were placed in the midst of limestone, the palm toddy were sprinkled on it. This process was repeated for 6 times and the SA nuts were washed and dried (21).

S2
2 SA nuts were kept in a white cotton cloth and tied at the top to make kizhi. This kizhi was kept in the mud pot and boiled in decoction of tamarind leaves, decoction of Butea monosperma, filtrate of cow dung mixed with water and Aloe vera juice separately (21).

S3
3 SA nuts were taken in a mud pot without removing the cotyledons. Palm toddy and curd prepared from the milk of red colour cow were mixed and SA nuts were immersed in the mixture for 9 days continuously (21).

S4
4 The cotyledons removed SA nuts were soaked in the kazhuneer (kaikuthalarisi soaked water) for 3 h. Then the SA nuts were soaked in buffalo milk for 3 h and were used for analysis after drying (24).

5
The cotyledons removed SA nuts were boiled in the filtrate of cow dung mixed in water for 3 h. The process was done for 7 times as said above. The above processed SA nuts were made into Kizhi and kept in puliilai kudineer (Tamarindus indica leaf decoction) and boiled for 3 h. The above processed SA nuts were immersed in cow's milk and kept for one day. The next day it was boiled for 3 h. Then they were processed with Aloe vera juice and tender coconut in the same manner. The above processed SA nuts were immersed in Aloe vera juice and kept for one day. Then it was boiled for 3 h(25).

S6
25ml water. The insoluble ashes were collected using ashless filter paper and washed with hot water and then transferred to the silica crucible, ignited the ashless filter paper for 15min at 600 o C. The silica crucible and residue were weighed until constant weight is attained. The weight of the watersoluble ash was determined by subtracting the weight of insoluble ash from the weight of total ash.

Acid insoluble Ash
The ash obtained from the above was boiled for 5 min with 25 ml 10%HCl.The insoluble ashes were filtered and collected in a crucible.Washed with hot water and ignited to constant weight. Percentage of acid insoluble ash was calculated.

Determination of Extractive Value Alcohol Soluble Extractive
Four grams of test drug were weighed and macerated with 100ml of absolute alcohol in a closed conical flask.The solution was shaken continuously for 6 h and allowed to stand and soak for 18 h. The solution was filtered and the filtrate was evaporated in a petri dish at 105 o C, then cooled and weighed. Percentage of alcohol soluble extractive value was calculated.

Water soluble Extractive
Four grams of test drug powder was weighed and macerated with 100 ml of water, in a closed conical flask. The resulting solution was shaken continuously for 18 h and allowed to stand and soak for 18 h and then filtered. 25 ml of the filtrate was evaporated to dryness in an evaporating dish, dried at 105 o C and weighed. Percentage of water soluble extractive value was calculated.

pH
10% solution of SA nuts was prepared in distilled water (w/v) and pH was determined by using digital pH meter.

Macroscopy of nuts
The dried fruits of Semecarpus anacardium are laterally flattened, drupaceous, dark brown, obliquely ovoidnut, surface smooth and sometimes shining with residual receptacle. The fruits of the six samples differed slightly in their dimensions. The S4 sample was biggest among the six samples measuring 2.8 to 3.5 cm in length and 2 to 2.5 cm in breadth. The dimensions and the surface morphology of the individual samples are given in Figure 1 and Table 2.  It was observed that the surface morphology of the nuts varied with the samples. Sample S6 had a nearly smooth surface followed by S1. Sample S2 had a charredlike surface may be due to the purification by treatment with lime. Samples S3 and S5 had nearly similar type of surfaces. In the sample S4 minute golden patches were visible which was absent in all the other five samples.

Microscopy
Microscopically the raw nut sample showed the fruit wall differentiated into epicarp, mesocarp and endocarp. Epicarp shows epidermis consisting of single layer of elongated lignified cells arranged radially covered above by a thin layer of prominent cuticle. In some places some of epidermal cells rupture along with the cuticle and resin drops is exuded out. Mesocarp consists of broad zone of parenchymatous cells arranged in numerous (>25) layers. The cells of the upper layers are bigger in size while the lower layer cells are smaller. Rosette crystals are seen randomly scattered. Fibro vascular bundles are also seen scattered. Some of the mesocarp cells get dissolved to form the lysogenic cavities filled with resin. Endocarp is differentiated into outer shorter and thinner layers of cells followed by prismatic layer of radially arranged very elongated thick walled cells. Below the endocarp S1 S2 cotyledons are seen containing numerous fixed oil cells and oil globules (Figure 2).

Figure 2. Microscopy of Semecarpus anacardium nut sample -S1
Cu -Cuticle; Ec -Epicarp; Mec-Mesocarp; Res -Resin canal; RC-Rosette crystal; Ve -Vessel Microscopically S2 showed charring of the cell wall, the contents of the cells such as resin content is not observed clearly ( Figure 3).

Figure 3. Microscopy of Semecarpus anacardium nut sample -S2
Microscopically S3 showed characters similar to that of raw nuts (Figure 4). Microscopically S4 showed characters similar to that of raw nuts ( Figure 5). Microscopically S5 showed characters similar to that of raw nuts ( Figure 6). Microscopically S6 showed characters similar to that of raw nuts (Figure 7).  The effect of the purifications processes performed on the drug was not evident as any change in the microscopical structures or content of the nut was not observed.
Powder of raw nuts (S1) showed characters as shown in the Figure9.

Figure 9. Powder microscopy of sample S1
Powder of S2 showed characters as shown in the Figure 10.   Powder of S5 showed characters as shown in the Figure 13.  Physico-chemical characterization showed differencesin values for loss on drying, total ash, water soluble ash, acid insoluble ash, water soluble extractive, alcohol soluble extractive and pH between samples S1 to S6 (Table3.)Sample S2 showed vast difference in all the parameters tested when compared to other samples.

Discussion
Macroscopic, microscopic and powder microscopic features of six samples of Semecarpus anacardium nuts purified by different Siddha methods have been studied. Slight difference in the texture of fruit wall was found among samples; the texture in case of sample S2 was totally different showing charred appearance as it is treated with limestone. Microscopically no remarkable differences were observed except sample S2; cell structure and content of sample S2 have been altered remarkably by the limestone treatment. The results of macro-microscopic observations matched with that reported earlier in the literature (28). There are about 5 types of calcium oxalate crystals present in plants in the following forms such as prisms, styloids, raphides, druses and crystal sand (29,30). Researchers reported that these crystals are not toxic component but they act as defense mechanism and protect the plant parts from foraging animals (31,32). The prismatic crystals and rosette crystals are found in these samples. The crystals are used for identification a raw herb under microscope (33).The quality and purity of the drug has been studied by the pharmacognosy characters of anatomy and cell inclusions in all the 6 samples (34) which has been proved by the reduction of anacardic acid in Semecarpus anacardium nuts after the above mentioned purification processes (35).
The Loss on drying which reveals the moisture content of all the samples were within the limits (5 to 8%) as per WHO (27). Low moisture content in the samples indicates a better stability and shelflife. An herbal sample free of moisture and can be stored for a long time without the microbial contamination. The acid insoluble ash in all samples except S2 represents lower inorganic matter and less contamination, also it plays important role in absorption in the gut (36). Higher limit of acid insoluble ash in sample S2 maybe due to the presence of lime stone involved in the purification process. High extractive value indicates higher content of secondary metabolites; the test is indication of absence of exhausted material, adulteration and substitution (37). High alcohol soluble extractive compared to water soluble extractive shows that Serankottai is less soluble in water because of its oily nature.But other ingredients which are used in formulations may change its solubility. Extractive values usually represent presence of many phytoconstituents which can be identified by HPTLC.The alcohol soluble extractive values indicated the presence of polar constituents like phenols, alkaloids, steroids, flavonoids while the water soluble fraction might be due to presence of glycosides. Water soluble extractive values observed shows that the glycoside presence as evidenced from phytochemical quantification analysis. Further chemical studies may give some clue on possible effects of treatments with different media.

Conclusion
According to Siddhars, even a poisonous drug can be converted to a life saving medicine. SA nuts after suthi process do not cause any allergy or untoward effect when it is consumed as prepared medicine (38).A preliminary study to evaluate the changes that occurred in the Semicarpus anacardium nuts has been documented in the samples before and after the purification process.This study may give an idea on the physicochemical and pharmacognostic parameters and their difference in raw and purified samples. Further studies can be done to document the possible chemical changes during Suthi process.