Some Problems Related to Quality Control

Heavy Metals in Ayurvedic Pills

Gulika (pills) are an important dosage form used in Ayurveda. Nearly 30 gulika are used in contemporary ayurvedic practice (Anonymous 2017b). Ayurvedic pills are prepared using a unique technology. Powders of herbs are mixed with various fluids like water, decoctions or juices of herbs, lemon juice, juice of pomegranate flowers or fruits, the latex of Jatropha curcas, cow milk, breast

FIGURE 4.3 Overlay of chromatograms of in-house formulation and marketed formulations of Arjuna curna. Reproduced with permission from Chitlange et al. 2009.

FIGURE 4.4 Overlay of chromatograms of isolated sapogenins, standardized in-house formulation and marketed formulations. Reproduced with permission from Chitlange et al. 2009.

FIGURE 4.5 H.P.T.L.C. plate of three batches of Pathyasadangam kvalha under U.V. 254 nm. Reproduced with permission from Abraham et al. 2018.

FIGURE 4.6 H.RT.L.C. plate of three batches of Pathyasadangam kvatha under 366 nm. Reproduced with permission from Abraham et al. 2018.

milk, cow urine, goat urine, castor oil, honey, clarified butter or sesame oil. The mass is ground on granite stone slabs using granite pestles. The grinding is carried out continuously for long periods, sometimes lasting even seven to 18 days (Vaidyan and Pillai 1985). Thereafter, the groundmass is rolled into pills of various sizes. Chemical transformations are bound to occur under such conditions, and these merit serious study.

Sebastian et al. (2015) carried out an extensive study of the heavy metal content of traditional ayurvedic medicines. The content of lead, arsenic, cadmium and mercury in 126 ayurvedic medicines manufactured by 32 companies was analyzed using inductively coupled plasma mass spectrometry (I.C.P.-M.S.). All the samples of kvdtha, ciirna, taila, ghrta, lihyam, asava, arista, dravakam and lipa analyzed in the study conformed to the limits for heavy metals set by the Government of India. However, unlike the results reported by Sebastian et al. (2015), many traditional ayurvedic pills were reported to contain high levels of heavy metals (Thomas and Kumar 2018).

A probable source of heavy metal contamination in ayurvedic pills may be the traditional method of preparing them. Granite is basically an igneous rock and contains mercury. Its mercury content is nearly twice that of crustal material (McElroy 2010). Granite also contains, in addition to other heavy metals, 0.2-13.8 ppm of arsenic, 0.003-0.18 ppm of cadmium and 6-30 ppm of lead (Cannon et al. 1978; Nagajyoti et al. 2010). It needs to be ascertained whether such a grinding method influences the heavy metal content of ayurvedic pills.

Souring of Ariṣṭa and Āsava

Ayurvedic medicine manufacturers often complain that arista and asava turn sour. Uncontrolled fermentation often results in acidity and off-flavors in the product. As the manufacture of arista and asava is very similar to that of making wine and other fermented beverages, the Ayurveda industry can learn from brewery technology. Breweries adopt various strategies to control the factors that cause spoilage.

Quality of Water

Water is the principal ingredient used in breweries. Water contributing directly as an ingredient of brewed products is called brewing water. Brewing water is treated or adjusted to achieve the correct composition relevant to the product being brewed. There are many contaminating micro-organisms present in water. Therefore, usually a few key indicators of the presence of both harmful and harmless species are selected for analysis. Routinely, water is examined for Escherichia coli as an indicator of fecal contamination. If E. coli is present, it can be assumed that there is a possibility of other pathogenic organisms being present. Only water conforming to microbiological and chemical specification is to be used in brewing (Taylor 2006).

Quality of Herbs

Vermouth is an aromatized and fortified wine popular in Russia, Poland and other European countries. It is basically white wine fortified and infused with a proprietary recipe of different herbs, barks, seeds and fruit peels. Vermouth is very similar to ayurvedic asava. Great care is taken in handling the herbs and spices, as their quality is affected by the harvesting and storage conditions. It is reported that the longer the dried products are stored before use, the poorer will be their flavor. Therefore, the manufacturers of vermouth ensure that the dried herbs and spices are used as fresh as possible (Panesar et al. 2009). A similar approach can be adopted in the manufacture of ayurvedic arista and asava as well. The quality of these products can be improved by using good quality herbs and jaggery devoid of microbial and chemical contamination.

Use of Appropriate Brewing Vessel

Micro-organisms attach to surfaces and develop biofilms (Donlan 2002). Biofilms resist sanitization during the washing process and allow bacteria to spread across the product (Srey et al. 2013). This is one of the major causes of spoilage of arista and asava. The threat of biofilms is apparent when wooden vats are used in the fermentation of arista and asava. It is difficult to disinfect wooden vats. Stainless steel tanks, on the other hand, are more hygienic and easier to clean. Moreover, biofilm production on stainless steel is less intense (Storgards 2000). Wooden vats require seasoning, and even after seasoning there are chances of evaporation and diffusion of the fermenting fluid through the minute pores of wooden vats, affecting the total yield of the product. Moreover, there is the possibility of the entry of free oxygen into the fermenting medium through the minute pores of wooden vats, as a result of which alcohol may be converted into acetic acid by oxidation. Such arista and asava become too sour in taste and become unfit for therapeutic purposes (Hiremath and Joshi 1991). Therefore, stainless steel brewing tanks need to be used instead of wooden vats.

Hygienic Design of Brewing Area

High care areas where critical operations are carried out are to be separated physically. Barriers need to be raised to prevent the entry of micro-organisms from raw materials, people, air or utensils (Walter 2006). Contamination during the brewing process is one of the most prevalent problems, and, therefore, the building should be planned and constructed meticulously.

Disinfection

Fermentation of arista and asava is a very sensitive process. Therefore, the fermentation room, brewing tanks and bottling equipment should be disinfected periodically. The aim of disinfection is to reduce the surface population of viable micro-organisms after cleaning and to prevent microbial growth on surfaces during the idle time (Storgards. 2000).

The brewing tanks, walls and floors should be cleaned thoroughly and disinfected before the start of operations. The common disinfectants used in breweries are hydrogen peroxide- peracetic acid, acid iodophores, quaternary ammonium compounds and halogenated carbonic acid (Storgards 2000). Manzano et al. (2011) provided an example of a cleaning and disinfection protocol. Fermentation vessels were rinsed with water at 75°C and cleaned with a 6.25% basic detergent solution at 85°C for 20 minutes. After cleaning, the tank was rinsed with cold water, cleaned with a 6.25% acid detergent solution in cold water for 20 minutes and then rinsed with cold water. Microbial contamination of arista and asava can be considerably reduced by this approach.

Use of Yeast

Ayurvedic arista and asava are brewed traditionally using flowers of Woodfordia fruticosa as a source of inoculum. Kroes et al. (1993) investigated the significance of W. fruticosa flowers in the preparation of Nimbarista. They noted that the flowers per se are not the source of alcohol-producing micro-organisms. But the flowers show considerable invertase activity, and this explains the alcohol content of this arista. However, the use of W. fruticosa flowers as a source of inoculum gives varying results. A study of the alcohol content of more than 500 commercial samples of ayurvedic arista and asava showed that their alcohol content varied from 2% to 12.88% (Figure 4.7) (Thomas et al. 2016). According to The Ayurvedic Pharmacopoeia of India, asava and arista should contain not less than 5% and not more than 10% alcohol (Anonymous 2008d). To maintain the required amount of alcohol, it is better to adopt fermentation technology employed in the making of wine, including the use of yeast to cause fermentation.

Temperature

Temperature has a general accelerating effect on fermentation. At higher temperatures (35°C) the carbon dioxide dissolved in the fermentation medium gets converted to compounds like fusel oils (isoamyl alcohol and phenyl ethanol) and higher alcohols like 1-propanol, 1-butanol, 2-butanol, isobutanol, isoamyl alcohol and 1-hexanol which cause liver disease (Lachenmeier et al. 2008). The maximum specific growth rate of yeast is known to be at 25°C (Torija et al. 2003; Sener et al. 2007). Therefore, it is desirable to keep fermentation temperatures of arista and asava around 25°C.

Improvised Fermentation

For maintaining the high quality of the arista or asava, all the ingredients used in their production should be subjected to physicochemical and microbiological analysis. Only ingredients that conform to the specification should be accepted for production. Deionized water alone is to be used in production. Instead of wooden vats, it is better to use stainless steel fermentation tanks.

FIGURE 4.7 Percentage of alcohol in commercial samples of asava and arista. Values in shaded areas conform to the pharmacopoeia range of alcohol. Reproduced from Thomas et al. 2016.

The fermentation tank should be housed in an aseptic, closed room maintained at a temperature of 25-30°C and relative humidity of 40-45% (Thomas et al. 2016).

The decoction of herbs or juices of herbs or fruits is to be prepared as instructed in the Sanskrit source of the formula. The specific gravity of the decoction or juice should be determined. Jaggery or honey is to be added to the decoction or juice. The specific gravity of water is 1, and when herbs are boiled in water, the resultant kvatha will have a slightly higher specific gravity. For example, Vara Asanddi kvatha has a specific gravity of 1.0185 (Ramachandra et al. 2012). When sugar is added to this kvatha, the specific gravity of the kvatha will increase by factor Y. Then specific gravity of the kvatha will be X + Y, where X is the specific gravity of kvatha. The required range of specific gravity, in respect to water, to yield 5-11% alcohol is 1 + 0.0388 (Ya) to 1 + 0.0799 (Yb). So increase in specific gravity is needed to get the desired percentage of alcohol = (X + Ya) to (X + Yb). An increase in 0.1° Brix increases specific gravity by 0.0004 and alcohol yield by 0.1% (Thomas et al. 2016).

Degrees Brix is the content of sugar in an aqueous solution of sugar. 1 g of sucrose dissolved in 100 g of water yields 1 Brix. Brix value is determined using a hydrometer or refractometer (Ribereau-Gayon et al. 2006). Following the Brix determination, the acidity of the fermentation medium is to be checked and adjusted to the desired value, as described above. As fermentation is to begin, a baseline analysis for reducing sugar, titratable acidity, pH, acetic acid and volatile acidity is to be conducted. This will help to track the progress of fermentation (Thomas et al. 2016).

Dried activated yeast (D.A.Y.) is used to cause fermentation of the medium. D.A.Y. is added to warm water (35-40°C), suspended well and added to the fermentation medium. A dose of 10-20 g of D. A.Y./100 liters is generally recommended (Ribereau-Gayon et al. 2006). The fermentation tank is to be left undisturbed for a specified period of time (Thomas et al. 2016).

Several methods are used to monitor fermentation. Important among them is the counting of yeasts. After diluting the fermentation medium, the total number of yeast cells is counted under the microscope using a Malassez cell. The total cells enumerated in this way include both “dead” yeast and “live” yeast. The counting of “viable cells” is a better way to differentiate between the “dead” and “live” cells. When the diluted fermentation medium is placed in a solid nutritive medium, the viable yeast cells form a microscopic cluster. The number of viable yeast cells is enumerated by counting the colonies formed on this medium after nearly four days (Ribereau-Gayon et al. 2006).

The rate of fermentation can also be tracked by estimating the amount of sugar consumed or the alcohol generated. As a relationship exists between the amount of alcohol generated and the initial concentration of sugar in the fermentation medium, the mass per unit volume (density) can give directly an approximate potential alcohol. The density and potential alcohol are usually marked on the stem of the hydrometer (Ribereau-Gayon et al. 2006).

The monitoring of tank temperature daily during fermentation is indispensable to keeping a tab on fermentation. The tank temperatures are different in different areas of the tank. The temperature is highest in the cap and lowest at the bottom of the tank. The temperature is usually taken after a pumping-over, which agitates the fermenting medium and homogenizes the tank temperature. Average tank temperature can be measured in this way (Ribereau-Gayon et al. 2006).

Cessation of fermentation is confirmed by the exact analysis of residual reducing sugar. Volatile acidity, acetic acid, titratable acidity and pH are measured. In addition to these tests, the arista or asava may be tested for the following parameters, like specific gravity at 20°C, total solids, alcohol content, methanol content, non-reducing sugar, T.L.C./H.RT.L.C. identification, heavy metals, microbial contamination, specific pathogens and pesticide residues (Lohar 2008). A flowchart of the proposed protocol for the brewing of asava and arista according to principles of modern brewery technology is presented in Figure 4.8.

By using yeast, the alcohol content of these products can be maintained within the legal limits. Alcohol acts as a preservative and extends the shelf-life of these arista and asava. Yeast-aided fermentation also extracts a wide range of compounds from the herbs, as the menstruum undergoes a gradient of rising alcohol levels (Hiremath and Joshi 1991).

A gas chromatographic study of 12 brands of Asvagandharistam sold in Sri Lanka revealed that in addition to ethanol, the products contained methanol, n-propanol, iso butanol, amyl alcohol and

isopropyl alcohol (Weerasooriya and Liyanage 2006). The manufacturing technology of arista and dsava, therefore, needs to be evaluated.

Irrational Substitution

Some instances of intentional addition of heavy metals to herbal formulae are also now known. An example is the addition of lead sulfide to Pusyanuga Сйгпат, indicated in dysfunctional uterine bleeding, piles, diarrhea and bloody stools. According to the formula described in Caraka Samhita, equal quantities of 25 herbs, including Arjuna (Terminalia arjuna) are to be powdered, mixed with honey and consumed (Sharma 1994a). The same formula is described in Cakradattam also (Sharma 1994b). In agreement with classical texts, the formula given in The Ayurvedic Formulary of India also recommends Arjuna (Anonymous 1978b). However, due to unknown reasons, several manufacturers in Kerala continue to substitute the bark of Arjuna with the similar-sounding mineral ahjana (lead sulfide) (Anonymous 2018b). A recent analysis of ten market samples of Pusyanuga Сйгпат revealed that six of them contained 4% of lead sulfide (Dr N.P. Damodaran, International Institute of Ayurveda, Coimbatore, personal communication). In light of the toxicity of lead (Pearce 2007; Flora et al. 2012), such irrational substitutions should be prohibited.