Preparation and chemical characteristics of Karunguruvai Khadi used in the traditional Siddha formulation of herbo-mineral-based medicine.

INTRODUCTION: Khadi is an extract that has been used in the preparation of Pooneeru (muppu) in Siddha medicine, but it has not been scientifically evaluated for modern medicinal purposes. The present study examines the preparation and chemical composition of Khadi.
MATERIALS AND METHODS: According to the method of the palm leaf literature, Karunguruvai and Samba paddy grains were selected for the preparation of Khadi. After removing the husk of the paddies, the cleaned Karunguruvai and Samba rice were processed according to the methods of Swami and Ramasamy Khon. The processed Khadi of Karunguruvai and Samba were analyzed for chemical constituents and were microbiologically assessed. The chemical composition of the paddy grains was also evaluated to compare with the Khadi constituents.
RESULTS: The major elements of the paddy grains of Karunguruvai and Samba were calcium (CaO) and sodium (Na2O). The predominant trace elements in the Samba rice were nickel and cadmium, whereas Karunguruvai rice contained arsenic, nickel, copper, and cadmium. Samba Khadi contained the trace elements mercury, chromium, lead, copper, and cobalt, whereas Karunguruvai Khadi had a high content of mercury, vanadium, arsenic, nickel, copper, cadmium, barium, and strontium. Karunguruvai Khadi also contained silicon dioxide (SiO2), aluminum oxide (Al2O3), iron oxide (FeO), manganese oxide (MnO), calcium oxide (CaO), and sodium carbonate (Na2O). By contrast, Samba Khadi contained only ferrous oxide (FeO), calcium oxide (CaO), sulfur dioxide (SO2), and carbon dioxide (CO2), which were chemical constituents of the Samba rice. DISCUSSION: The composition of Karunguruvai rice was compared with Samba rice and it is identified the absence of heavy metals in Samba as well as Karunguruvai rice. Both of rice powders have more or less similar chemical compounds except phosphorous content. Karunguruvai rice possesses more CaO than Samba rice. The bacterial and fungal activities were assessed during the different stages of Khadi preparation-no activities were found in any form of the Khadi. This suggests that Khadi may have a preventive effect against fungal and bacterial infections.
CONCLUSION: The result of the chemical analysis of Khadi extracts showed that Khadi prepared from the Karunguruvai paddy grains (rice) was the best base solvent for ion exchange in the preparation of muppu than Samba Khadi. The analysis of the composition of these two types of Karunguruvai Khadi show that it is a good solvent for the elimination of heavy metals and for the enrichment of elements in Pooneeru powder (muppu), the chuurnam of Siddha drugs. These characteristics enhance the therapeutic potential and safety of the drugs for healing chronic diseases.

Introduction

Siddha is a traditional medical system of India. It is of Tamil origin and has its origins in the Tamil language. The Siddha medicine system use metals and minerals predominantly for treating varieties of joint diseases, skin diseases, liver problems, and urinary tract infections. Within the system, food is considered the ideal treatment. Rice (Oryza sativa L.) forms an essential component of the diet due to its nutritional content, including carbohydrates, proteins, and minerals, which increase the immunity of the human body. Rice (husk-removed paddy grains) is also known as “arisi” in Tamil and is one of the earliest known staples of the diet in Tamil history. In insular south-east Asia, the Austronesian terms “padi” and “paray” for rice and “bras” or “beras” for milled rice predominate. In Tamil Nadu, the Cauvery river basin has been renowned as the “rice granary of South India.” There has been approximately 400 varieties of rice in vogue since the Kingdom Era. The varieties were clearly distinguishable and given names according to color, shape, size, and appearance, either as an individual trait or in combination with other characters. Fermentation of Monascus fungi on steamed monascal rice (Red Koji in Korea or Hon-Chi (紅麴 hóng qū) in Mandarin) has been used for the preparation of traditional medicine, food colorant, wines, and other fermented food in oriental countries for more than 600 years.5, 6, 7

The preserved mineral extracts from plants are used to heal the body from decomposition. Muppu is a mineral extract, which has a significant role in Siddha medicine system. Three ingredients are usually added to the preparation of medicinal muppu—sodium carbonate (Pooneeru), rock salt (Kaluppu), and Vediuppu (potassium nitrate). Throughout Tamil Nadu, various types of muppu extracts have been reported: vaidya muppu; the medicinal form vada muppu, which is used in south Indian alchemy; and yoga muppu, which is used exclusively as an aspirant and has other rejuvenating and antioxidant benefits. In a previous study, muppu was prepared from three types of salts collected from different parts of the southern Tamil Nadu as per the palm leaf literature description (Kandarnadi Vaakiyam—Ancient Tamil Siddha medicinal notes written on the palm leaf) for measurement of the hepatoprotective potential of muppu in rats. The results showed that the muppu extract had significant hepatoprotective properties.

Khadi is a fermented liquid consists of various chemical compounds which extracted from the boiled rice of Karunguruvai and Samba paddy grains. The fermentation process of boiled rice material is carried out based on the traditional method for preparation of the Khadi extract. The base change properties of Khadi extract is important in the Pooneeru preparation and it is used as base exchange solution in preparation of Siddha drugs. There are several steps involved in Khadi preparation. Khadi is an important solvent/mineral water used in Siddha medicine. Unfortunately, Siddha doctors forget its importance due to a lack of practical knowledge. Muppu is the crown of Siddha medicine. In the preparation of muppu, Khadi has a vital role. Gunapadam is the branch of Siddha, the healing heritage, which deals with the processing of stony, hard, macro, and highly toxic metals or minerals to explore their therapeutic efficacy and safety. It is important to explore the therapeutic claims of Siddha medicine in a scientific manner. Therefore, the Khadi preparation was chosen for the present work. Khadi is used in muppu preparation (Pooneeru) as a base-exchangeable medium. However, many researchers and medical practitioners use Khadi in very different forms (up to 80). There are numerous names for Khadi, which are confusing and lead to misunderstanding; many products have no relation to Khadi at all. Those who successfully handle Khadi enjoy the benefits of using it and establish themselves as very successful Siddha medical practitioners. However, there has been no research on the methods of preparation of Khadi and its characteristics have not been published. Therefore, the present study aims to examine the traditional methods of preparation of Karunguruvai and Samba Khadi with modern scientific analysis in order to characterize its chemical and microbial properties.

Materials and Methods

Selection of raw materials

Khadi is an extract from specific paddy grains—Karunguruvai (black-colored rice) and Samba (red-colored rice) cultivated in the Cauvery delta region, Tamil Nadu, India (Fig 1). Karunguruvai rice has more fibrous bran than Samba rice. It has nearly 5% amylase content and can influence gel consistency at gelatinization temperature. Therefore, Karunguruvai paddy grains are widely used in Siddha for Khadi preparation. Karunguruvai and Samba rice grains are shown in Fig. 2.

Fig. 1

Matured paddy plants: (A) Karunguruvai and (B) Samba.

Fig. 2

Harvested paddy grains of (A) Karunguruvai and (B) Samba. Cleaned paddy grains (rice) after removing husk of (C) Karunguruvai and (D) Samba.

Preparation of Khadi

Karunguruvai and Samba rice were processed by removing the husks, stones, mildew, and immature grains. Then, according to the methods reported by Swami and Ramasamy Khon,12, 13 the Karunguruvai rice was kept on a new mud pot and water was added to the grains in a ratio of 1:3 and boiled without decanting the excess water. The boiled rice was spread evenly over a palm leaf mat and kept in a cool place (Fig. 3).

Fig. 3

Boiled (cooked) Karunguruvai rice.

For the preparation of Khadi, a new mud pot was selected and its outer surface was coated with calcium hydroxide, prepared out of kankar lime (CaO). The inner surface of the pot was exposed with the fumes of sambirani (styrex benzoin) and kept closed. The pot was kept under bright sunlight for simple drying for 8 hours. The boiled Karunguruvai rice was transferred to the prepared pot with boiled cooled water in a ratio of 1:7, and stirred well for 30 minutes. Then the mouth of the pot was closed with a clean cloth to avoid contamination. The upper edge and mouth of the pot were swiped with a cloth (3 cm in width) dipped in castor oil to protect the pot from insects. The pot with the Karunguruvai rice and fresh water was kept in sunlight for 168 hours. Then, the pot with the boiled rice was kept in a safe place and covered with a lid to protect it from rainwater and dust (Fig. 4A–C).

Fig. 4

Mud pots used for the fermentation process: (A) pot coated with calcium hydroxide; (B) pots placed in the fumes of sambarani (styrex benzoin); (C) pot with lid to protect from rainwater and other atmospheric contaminants.

After 168 hours of drying, the contents of the pot were transferred to a new pot, and the same process was repeated. In the meantime, the original pot was reprocessed for further use. After 168 hours of drying was completed for the second pot, the mixture was brought to the first pot and the same procedure was repeated again. While changing the pots, necessary caution was exercised to maintain the volume of the earlier pot; if there was any reduction in the volume, additional boiled and cooled fresh water was added. Similarly, each stage of Khadi preparation is repeated over a period of six months to understand the physico-chemical changes. During this process, a change in taste was noted from month to month; however, there was no change in taste, color, or pH value after six months. It is this Khadi that has fermented for 6 months that is used for the preparation of traditional medicines.

Preparation of culture medium for bacterial and fungal growth testing

Traditionally, the control of microorganisms in any food material has been demonstrated by microbiological testing of samples at different stages of the process and the end-product. It is essential to understand the nature of potential hazards that may be presented by the raw materials. Microbiological testing is considered essential by traditional drug manufacturers and buyers in India to assess the safety of Khadi for use in drug formulations in terms of content of microorganisms (bacterial and fungal growth). Ideally, they should give a description of the drug, indicating key processing features and conditions under which the Khadi materials should be stored and used. Such factors significantly influence the content and validity of the microbiological analysis.

In this study, the three types of culture media used for bacterial growth testing were the nutrient agar plate (NAP), blood agar plate (BAP), and MacConkey's (MC) agar plate; Sabouraud agar plate (SAP) was used as the culture media for fungal growth testing (Fig. 5A and B). The preparation of bacterial culture should provide similar environmental and nutritional conditions that exist in its natural habitat. Hence, an artificial culture medium must provide all the nutritional components include water, agar, peptone, casein hydrolysate, meat extract, yeast extract, and malt extract, etc. The chemicals and nutritional components used for preparation of culture media are purchased from Himedia Chemical Company, Mumbai, India. For bacterial growth testing assays, the culture media were prepared using the following ingredients: peptone (5 g), sodium chloride (5 g), beef extract (1.5 g), yeast extract (1.5 g), and agar (15 g); these ingredients were then diluted using 1000 mL of distilled water and poured into a flat container. For fungal culture, the Sabouraud agar plate media was prepared using the following ingredients: meat and casein protein mixture (10 g), glucose (40 g), agar (15 g), Sabouraud mixture (26 g); the ingredients were mixed with distilled water (1 L) at a pH of approximately 5.6 ± 0.2. Then, drops of the Khadi extracts were streaked onto both nutrient agar plates and Sabouraud agar plates, sealed, and stored at room temperature (15–20°C).

Fig. 5

Culture media of (A) bacterial and (B) fungal growth tests of Khadi extracts.

Chemical analysis of Khadi

Filtered Khadi samples were analyzed using the chemicals purchased from Sigma Chemical Company, USA. The chemicals employed were of analytical grade for Hg, As, Cr, V, Pb, Ni, Cu, Cd, Co, and Ba using HG-ET-AAS (Hydride Generation Electro Thermal Atomic Absorption Spectrometer – Perkin–Elmer Zeeman 3030). Calibrations were made by standard additions by adopting Susana Santos et al. 2013. Similarly, the trace elements for paddy grains were determined after dry mineralization of both dry samples and infusion evaporated to dryness prior to HG-ET-AAS measurements. Samples were ashed in an electric furnace at 540°C with a gradual increase of temperature and subsequent dissolution of residues in HCl.14, 15

Results

The following observations were recorded during the preparation of Khadi:

At the beginning of the 1st month, the Khadi in the pot had no taste, had a brown color, no fermentation, and the pH was neutral.

At the end of the 1st month, the had a sour taste. It showed foaming fermentation at the upper surface. However, small bubbles were seen at the bottom and the pH was becoming alkaline.

At the end of the 2nd month, the sour taste gradually increased day by day. A large amount of foam appeared due to the fermentation effect on the surface. The rice in the mixture started to dissolve in the water. At the close of this month, the sour taste changed to bitter and the pH became strongly alkaline.

At the close of the 3rd month, whatever foamy bubbles appeared in the last month reduced during this period. The bitter mixed taste also changed gradually. More rice was found to have been dissolved. The liquid mixture in the 3rd month was tasted for Thuvarppu, to measure its astringency.

At the close of the 4th month, a complete disappearance of foamy bubbles was found on the surface of the Khadi liquid. The light brown color was slowly changing to colorless. The rice in the liquid was largely dissolved. The Thuvarppu taste was much enhanced at the close of the 4th month. The liquid was now becoming vuarppu (saltish taste).

At the close of the 5th month, there was no foam in the liquid. The rice was completely dissolved in the liquid and it became light brown in color. The salty taste became pungent.

At the close of the 6th month, it should be noted that the water was highly transparent. The taste of the liquid became slowly sweeter at the beginning of this month. However, by the end of the 6th month, the Khadi became tasteless, colorless, and had no smell. The Khadi was preserved in a jar, and exposed to the fumes of sambirani (styrex benzoin).

The details of the fermentation process are shown in Table 1.

Table 1

The fermentation processes in Khadi preparation.

Sl. No.	Period of observation	Total number of days	Change of color	Change of taste	Change in pH
1	At the beginning of 1st month	1	Brown	No taste	Neutral
1	At the close of the 1st month	30	Brown	Sour taste	Alkaline
2	At the close of the 2nd month	60	Brown	Bitter taste	High alkaline
3	At the close of the 3rd month	90	Light brown	Astringency	Less alkaline
4	At the close of the 4th month	120	Pinkish red	Saltish	Alkaline
5	At the close of the 5th month	150	Light pink	Pungency	Alkaline
6	At the beginning of 6th month	180	Very light pink	Sweetish	Acidity
7	At the close of the 6th month	180	No color	No taste	Neutral

Chemical composition of Khadi

In order to determine the application of Khadi as an ideal solvent, chemical analysis was carried out to quantify the trace elements and major elements of Khadi as well as paddy grains to evaluate permissible limits and toxicity. Trace elements play different positive and negative roles in humans, animals, and plants. Trace elements such as As, Cu, Fe, and Ni are considered essential at low concentrations but are toxic at high levels,16, 17, 18 whereas Al, Cd, Pb, and Hg have no known significant biological functions in Khadi. The ancient Tamil Saints (Siddhars) used Karunguruvai rather than Samba rice because of the high content of CaO and K2O and the fibrous layer containing medicinal properties (Table 2, Table 3). After completion of 6-month fermentation, the fermented Khadi of both types of rice powders, Samba and Karunguruvai, were chemically analyzed (Table 4, Table 5). Khadi was found to be without any taste, odor, or color after 6 months. The pH at every stage of the preparation remained stable due to the nonavailability of alcohol.

Table 2

Major elements in cleaned rice of Karunguruvai and Samba paddy grains.

Sl. No.	Sample description	Major elements
SiO2(%)	Al2O3(%)	TiO2(%)	FeO(mg/L)	MnO(mg/L)	CaO(%)	K2O(mg/L)	Na2O(%)	P2O5(%)	SO2(%)	CO2(%)
1	Samba rice	ND	ND	ND	ND	ND	413	ND	1250	ND	ND	ND
2	Karunguruvai rice	ND	92	ND	ND	ND	470	16	1250	ND	ND	ND

ND = not detectable.

Table 3

Trace elements in cleaned rice of Karunguruvai and Samba paddy grains.

Sl. No.	Sample description	Trace elements (in mg/L)
Hg	As	Cr	V	Pb	Ni	Cu	Cd	Co	Ba	Sr
1	Samba rice	ND	ND	ND	ND	ND	413	ND	1250	ND	ND	ND
2	Karunguruvai rice	ND	92	ND	ND	ND	470	16	1250	ND	ND	ND

ND = not detectable.

Table 4

Major elements in Khadi (fermented liquid) of Karunguruvai and Samba paddy grains.

Sl. No.	Sample description	Major elements
SiO2(%)	Al2O3(%)	TiO2(%)	FeO(mg/L)	MnO(mg/L)	CaO(%)	K2O(mg/L)	Na2O(%)	P2O5(%)	SO2(%)	CO2(%)
S3	Samba Khadi	–	–	–	0.36	0.01	50.67	0.02	0.86	–	2.21	45.88
S4	Karungurvai Khadi	4.46	12.43	0.27	1.60	0.01	20.10	0.02	0.17	ND	4.00	57.40

ND = not detectable.

Table 5

Trace elements in Khadi (fermented liquid) of Karunguruvai and Samba paddy grains.

Sl. No.	Sample description	Trace elements (in mg/L)
Hg	As	Cr	V	Pb	Ni	Cu	CD	Co	Ba	Sr
1	Samba Khadi	2	ND	20	ND	525	ND	5	ND	2700	ND	ND
2	Karunguruvai Khadi	121	42	ND	2750	ND	260	8	500	ND	151	85

Table 2, Table 3 show the major and trace elements in both Samba and Karunguruvai rice. Both had similar contents, but the Karunguruvai contained additional Al2O3, As, Cu, and K2O. This result demonstrated the protective layer of inert materials in the paddy grain. The presence of Al, As, Cu, and Cd in Karunguruvai rice is considered superior by Siddhars in the preparation of Khadi.

Table 4, Table 5 show the major and trace elements in the Khadi solvent of both Samba and Karunguruvai rice. It was observed that Samba Khadi contained significant amounts of lead, chromium, copper, mercury, and cobalt. However, Khadi prepared from the Karunguruvai rice showed the presence of mercury, arsenic, vanadium, nickel, and cadmium.

As for as the major oxides, Samba Khadi contained FeO, MnO, CaO, K2O, Na2O, SO2, and CO2, whereas Karunguruvai Khadi contained SiO2, Al2O3, TiO2, FeO, MnO, CaO, K2O, Na2O, SO2, and CO2; the details are shown in Table 5. The gelatinization temperature was determined using the Patindol and Wang method and viscosity was determined according to the AACC (American Association of Cereal Chemists) method. The gelatinization temperature for both Samba and Karunguruvai Khadi varied between 75°C and 80°C; similarly, the viscosity ranged from 300 to 8000 Ns/m2.

An ethyl alcohol test was carried out for the Khadi and no yellow precipitate of iodoform was found. Fig. 6A and B show the ethyl alcohol test using iodoform and the starch test using an iodine solution, respectively. On the starch test, the absence of a violet color confirmed the absence of polysaccharides such as starch (Table 6, Table 7).

Fig. 6

(A) Ethyl alcohol test using iodoform; (B) starch test using iodine solution.

Table 6

Growth test of bacterial species in Khadi (fermented solution) of Karunguruvai paddy grains.

Sl. No	Bacterial species	Culture media for bacterial growth
NAP	MC	BAP
1.	Eschericia coli	NG	NG	NG
2.	Streptococcus aureus	NG	NG	NG
3.	Klebsiella pneumoniae	NG	NG	NG
4.	Salmonella typhi	NG	NG	NG

NG = no growth.

Table 7

Growth test of fungal species in Khadi (fermented solution) of Karunguruvai paddy grains.

Sl. No	Fungal species	Culture media (Sabouraud agar plate)
1.	Candida albicans	NG
2.	Aspergillus fumigatus	NG

NG = no growth.

Further, the screening of important physico-chemical components, total phenolics, DPPH and ABTS radical screening are currently in progress.

Discussion

The composition of Karunguruvai rice was compared with Samba rice, and we found the absence of heavy metals in Samba as well as Karunguruvai rice. Compositionally, except phosphorous, in all other aspects both rice powders were more or less similar. Karunguruvai rice possesses more CaO than Samba rice.

At the different stages of Khadi preparation, the influence of bacterial and fungal contamination can be expected, however such bacterial and fungal impacts were not found in this analysis. This suggests that Khadi may have a preventive effect against fungal and bacterial infections. In view of the contrasting characteristics, Siddhars must have preferred Karunguruvai to Samba. In the Karunguruvai Khadi extract, major oxides and elements in inorganic form such as vanadium, cadmium, barium, strontium, and mercury were found at the final stage of processing. Both are high in carbohydrates but the Khadi of Karunguruvai remains stable without many changes from rice powder to Khadi except marginal reductions in iron, cadmium, nickel, arsenic, SiO2, CaO, and SO2; therefore, it is chosen as the best solvent by Siddhars. Further, the nature of chemical composition of both rice materials is not easily attacked during the fermentation processes. Instead, it gains many trace elements by absorption. In other words, the Khadi is capable of absorbing and retaining important heavy metals within its structure; otherwise transformation processes in the enrichment of major and trace elements would occur due to the tendency of starch in the beginning of the process to undergo retrogradation and viscosity. The water uptake ratio was higher for Karunguruvai than Samba during Khadi preparation. Knowledge of Khadi chemical composition helps to elucidate the bioconversion changes of Pooneeru (muppu) in Siddha drugs. The component should be subject to further studies to verify its quality. During the stages of fermentation, the Khadi was tested for bacterial and fungal growth; no infection or microbial growth was evident. The chemical aspects of Khadi explain the potency of muppu, and its benefits for potential drugs and its applications in alchemy.

Conclusion

Karunkuruvai and Samba paddy grains have been chemically analyzed to understand the advantage of Khadi. Among them, Karunkuruvai Khadi has higher base exchange characteristics than Samba Khadi. Therefore, Karunkuruvai Khadi is recommended for use in the preparation of Pooneeru chuurnam (muppu) in Siddha drugs. Karunguruvai Khadi was prepared according to the literature, and at every stage of fermentation, the difference in taste was studied. After 6 months of continuous fermentation, we can confirm that Khadi was odorless, colorless, and tasteless.

Khadi extract is a good solvent in the preparation of Pooneeru chuurnam (muppu) due to its enrichment of elements. This property is achieved depending on the literary skills of the Siddha modern practitioner. Knowledge of the traditional practices of Siddhars in Khadi preparation can sustain its modern usage. This paper provides a modest comprehensive procedure of Khadi preparation for Siddha medicinal formulation to enhance the therapeutic potential and safety of the drugs in Pooneeru chuurnam (muppu) for healing chronic diseases. More work is required to understand the transformation process of elements and other properties at each stage of Khadi preparation.

Conflicts of interest

All authors declare no conflicts of interest.