Physiochemical and Acute Toxicity Analysis of a Siddha Herbo-Mineral Formulation – Pachai Karpoora Vadagam (PKV)

 

Dr. Shunmugaram S^*

Research Officer (S), Siddha Regional Research Institute, Puducherry.

 

ABSTRACT:

Siddha system of medicine is one of the traditional systems of medicine which belongs to southern part of India. It widely uses herbs, metals and minerals as raw drugs for medicine preparation. The medicine which possesses metals and minerals are better to undergo physiochemical and toxicological analyses that help us to prove our drugs are nontoxic in nature. Pachai Karpoora Vadagam (PKV) is a Siddha Herbo-mineral formulation, prepared as per the Siddha literature Gunapadam Thaathu Seeva Vagupu. The phytochemical and physiochemical analysis like colour, odour, solubility, ash values and pH analysis were analyzed. The acute toxicity study was done in Swiss albino rats. The physiochemical of PKV revealed that all parameters were within normal range. The toxicity study did not exhibit any mortality in rats. No weight reduction and behavioral changes was noted. All internal organs were found to be normal in necropsy. Hence the Siddha formulation PKV is safe for human usage.

 

 

 

1. INTRODUCTION:

Siddha system of medicine is one of the ancient systems contemporaneous with those of submerged lands, Egyptian, Mesopotamian, Chinese and Grecian medicines. The unique nature of this system is, its continuous service to humanity for more than 5000 years in combating diseases and in maintaining its physical, mental and moral health while many of its contemporaries had completed their course. The word ‘Siddha’ means establishing truth. The pharmacopoeia of Siddha system contained large number of plants, metals, minerals and animal products. The purification, combinations, measurement and the methods of preparations are given with minute precision in Siddha literature.¹ Pachai Karpooram (Borneo Camphor) is one among the Kaara Saaram (Minerals) which is widely used in Siddha preparation for many years.

 

The drug Pachai Karpoora Vadagam (PKV) is a herbo-mineral preparation which consist single mineral Pachai Karpooram and 4 herbs. It has high therapeutic value. It is used to treating the diseases like Upper respiratory tract infection, Arthritis etc.² Standardization of Siddha mineral drugs in order to assess the quality was based on the concentration of their physical, chemical, phytochemical standardization. Hence the purification process, may reduce the toxic nature of the drug it is mandate now a days to ensure the safety of the finished product by conducting toxicity studies according to standard guidelines. It should ensure the safety and efficacy of the classical text formulation before human usage.

 

2. MATERIALS AND METHODS:

Pachai Karpoora Vadagam has selected from the Siddha literature “Gunapadam Thaathu Seeva Vagupu” page no.418.

 

2.1 Ingredients²:

The test drug PKV contains Pachai Karpooram (Borneo Camphor) 35 grams, Santhana Thool (Powder of Santalum album Linn.) 70 grams, Elam (Elettaria cardamomum Matan.) 105 grams and Arathai (Alpinia officianarum Hance.) 140 grams. The raw drugs were purchased from authorized country drug shop and Thulasi leaves (Ocimum sanctum Linn.) were collected from the herbal garden of National Institute of Siddha. The herbs and mineral were authenticated by the HOD, Department of Medicinal Botany, National Institute of Siddha and HOD, Department of Chemistry, SCRI, Chennai respectively. The drugs were purified as per the methods given in Siddha literature.

 

Table 1. Purification of Ingredients ^{3, 4}

Pachai karpooram	The camphor was Powdered and boiled with ghee 7 times. Fresh ghee was used each time.
Santhana Thool	The heart wood was taken out and powdered.
Elam	The whole fruit was roasted until it became golden colour with pungent smell.
Arathai	The outer membrane of the rhizome was peeled out then cut into small pieces and dried.
Thulasi leaves were cleaned with sterile cloth, removed dusts and waste parts of leaves.

 

 

Pachai Karpooram                                     Elakkai

 

 

Arathai                                                   Santhana Thool

Fig 2. Ingredients of Pachaikarpoora Vadagam

 

2.2   Preparation of the Trial Drug:

The purified raw materials were crushed, powdered and mixed well except camphor. Then the powder and camphor ground in kalvam with thulasi juice for 3 hours and made in to (Chundaikaai alavu) turkey berry sized pills (500mg) and dried in the shade then kept in labeled airtight container.

 

 

Fig 2. Prepared Pachaikarpoora Vadagam

 

3. Physicochemical Analysis⁵:

3.1.  Physical characterization of Drug:

a.      Colour Examination:

Ten tablets were taken in a watch glass and positioned against white back ground in bright white light. The colour of PKV was observed by naked eye.

 

b.    Odour Examination:

Ten numbers of tablets were smelled individually. The time interval between two tablets was two minutes to overturn the effect of previous smelling.

 

c.    Solubility:

1gm of drug was taken in a dry test tube and shaken well with distilled water. A small quantity of the drug was shaken well with Con.HCl and then Con.H₂SO₄. Test drug Solubility was observed.

 

d.    pH Value:

The drug solution was prepared with proper reagents and diluting with freshly boiled and cooled water. The pH value of the drug was determined potentiometrically by means of the glass, electrode and a suitable pH meter.

 

e.    Total Ash:

Two grams of grounded air dried powder of PKV was accurately weighed in a previously ignited and tarred silica crucible. The drug was gradually ignited by raising the temperature up to 450⁰c until the drug became white. The drug was cooled in desiccators and weighed. The percentage of total ash was calculated with reference to air dried drug.

 

f.     Acid Insoluble Ash:

The ash was boiled with 25ml of diluted hydrochloric acid for 5 minutes, the insoluble matter was collected on an ash less filter paper, washed with hot water, ignited cooled in a desiccator, and weighed. The percentage of acid insoluble ash calculated with reference to the air-dried drug.

 

 

g.    Loss of drying (Determination of moisture content):

Loss on drying is the loss in percentage w/w resulting from water and volatile matter of any kind that can be driven off under a specified condition. A glass stopper, shallow weighing bottle was weighed accurately, 10 gms of the drug was transferred and distributed evenly. The bottle was covered and weighed then placed in the drying chamber. The drug was then dried at 105⁰c for a specific period of time, and the bottle was removed from the chamber and allowed to cool at room temperature in desiccators before weighing.

The results of physiochemical analysis were given in table 3.

 

4. Preliminary Qualitative Phytochemical test:

5gm of drug was weighed accurately and placed in a 250ml clean beaker and added with 50ml of distilled water. It was boiled well for about 10 minutes, then cooled and filtered in a 100ml volumetric flask and made up to 100ml with distilled water. The phytochemical analysis of PKV was given in table 5.

 

5. Acute Toxicity Study⁶:

5.1.  Principle:

Acute toxicity of PKV was carried out in female Wistar albino rats dosed in a stepwise procedure as per OECD guidelines 423.

 

5.2.  Test Animals:

Animals were obtained from TANUVAS Madhavaram, Chennai and stocked at Animal House of National Institute of Siddha, Chennai. All the animals were kept under standard environmental condition (22°C ±3°).The animals had free access to water and rodent pelleted food (Nutrilab rodent, India).The principles of laboratory animals were followed and the proper protocol for animal study was approved by the Institutional Animal Ethics Committee and the study no is (NIS/IAEC/7/2013/I/24).

 

5.3. MATERIALS AND METHODS:

Animal Species	Wister albino rat
Sex	Female
Age/weight	6-8 weeks/150-175gms
Acclimatization Period	7 days prior to dosing
Housing	Individually in polyurethane cages
Identification	Animals will be kept in individual cages and numbered

 

Route of administration:

Oral route

 

Test substance:

Pachai Karpoora Vadagam (PKV)

 

Vehicle:

Hot water.

 

Administration of doses:

PKV was suspended in standard quantity of distilled water with uniform mixing and it was administered to the groups in a single dose through oral route. The control group was received equal volume of vehicle (Distilled water). The animals were weighed before giving the drug.

 

Table 2. Dose calculation according to animal body weight

S.No	Groups	No of rats and sex
1	Group I: Control (hot water)	6/f
2	Group II: 50mg/kg	6/f
3	Group II: 300mg/kg	6/f
4	Group II: 2000mg/kg	6/f

 

Observations^7,8:

Observations were made and recorded systematically and continuously observed as per the guidelines after drug administration. The animals were monitored for behavioral parameters like,

1. Awareness: Alertness, Visual placing, Stereotype, Passivity

2. Mood: Grooming, Restlessness, Irritability, Fearfulness

3. Motor activity: Spontaneous activity, Reactivity, Touch response, Pain response.

 

Animals were observed for mortality, body weight and behavioral changes for 14 days. At the end of the 14th day all animals were sacrificed and gross necropsy was done.

 

6.    RESULTS:

Animals treated with different doses of PKV did not exhibit any mortality in rats. No behavioral changes were noted for the first 4 hours and for the next 24 hours and throughout the study period of 14 days. No weight reduction was noted before and after the administration of test drug.  Reflexes were found to be normal before and after the study. All other observations were found to be normal before and after the study. No significant abnormality was noted during acute toxicity study. In gross necropsy, the organs of the animal such as, Liver, Heart, Lungs, Pancreas, Spleen, Stomach, Intestine, Kidney, Urinary bladder, Uterus appeared normal.

 

Table 3. Physiochemical Analysis of Pachai Karpoora Vadagam

S. No	Parameters	Results
1. Organoleptic Characters
	a.       Colour	Dark brown
	b.       Odour	Pleasant mild camphor odour
	c.       Sense of touch	Harden
	d.       Appearance	Rounded
	e.       Taste	Little bitter, Sour.
2.	pH	6.02
3.	Loss on drying @ 1050C	5.36%
4.	Total Ash	16.42%
5.	Acid insoluble Ash	3.50%
6.	Water Soluble Ash	4.43%

 

Table 4. Phytochemical Analysis of Pachai Karpoora Vadagam

S.No	Constituents	Inference
1.	Alkaloids	Present
2.	Phosphate	Present
3.	Ammonium	Present
4.	Tannic Acid	Present

 

7.    DISCUSSION:

Solubility is one of the important parameters to attain desired concentration of drug in systemic circulation for necessary pharmacological response. The bioavailability of drug in oral route depends on numerous factors including aqueous (or) lipid solubility, drug permeability, gastrointestinal motility etc. Solubility of the drug was high in water medium. So drug was easily absorbable in the gut.

 

PKV was slightly acidic in nature (6.02). The acidic drugs were easily absorbable in the stomach. So, the pharmacological action of the drug will be start when the drug reached at stomach.

 

The percentage of loss on drying @ 105⁰C was 5.36%. It was within acceptable range thus implied the drug contained low moisture and it can be stored for the period that given in Siddha literature.

 

Ash values:

The ash test is the effective parameter to determine the degree of purity of the preparation. It’s used to measure the amount of residual. A high ash value may indicate the contamination, adulteration, substitution, or carelessness in preparing the drug.  The total Ash value of PKV was 16.42% which correlate with the inorganic contents Phosphate, Ammonium and Tannic acid through the phytochemical analysis.

 

The Acid-Insoluble Ash limit is used to estimate the amount of ash insoluble in diluted hydrochloric acid which denotes the amount of siliceous matter present in the drug. The quality of the drug is better when the Acid-Insoluble Ash value is low. PKV showed a minimum percentage of Acid Insoluble Ash.

 

Preliminary Phytochemical analysis of the study drug reveals the presence of Alkaloids, Phosphate, Ammonium and Tannic acid. Alkaloids possess the anti-oxidant, analgesic, anti-inflammatory, anxiolytic, antidepressant, hepatoprotective, antiseptic, stomachic, anti-viral and anti-herpetic activities. The tannic acids possess the analgesic, anti-inflammatory, anti-microbial and anti-oxidant activities^9,10. The presence of phosphate reveals it help to treat bone and renal disorders. The presence and activities of alkaloids and tannic acid revealed that the drug may reduce the level of inflammation, pain intension and increase rejuvenation process of the diseases that prescribed for PKV in Siddha literature.

 

8.    CONCLUSION:

The phytochemical analysis results showed the relationship with the literature survey of the PKV which gives additional support to its clinical usage in clinical trial of Osteoarthritis as antioxidant, analgesic and anti-inflammatory action. The physiochemical analysis of PKV showed that all parameters were within normal range.  The acute toxicity analysis of PKV up to 2000 mg/kg body weight didn’t exhibit any toxicity. The above studies revealed that the prepared drug Pachai Karpoora Vadagam is in standard quality and safe for human usage.

 

9.    ACKNOWLEDGMENT:

I express my sincere gratitude to Prof. Dr. K. Manickavasakam, Director, NIS, Chennai for his valuable guidance and encouragement in this study. I express my sincere thanks to Chairman and Members of Institutional Animal Ethical Committee (IAEC) of NIS, Dr. N. J. Muthukumar, Associate Professor and HOD, Faculties of Department of Sirappu Maruthuvum, Dr. P. Aravind, Asst. Prof, (Medicinal Botany) and Faculties of Department of Gunapadam, NIS, Chennai for their guidance and co-operation.

 

10. REFERENCES:

1.     Kandaswamy Pillai N, History of Siddha Medicine, Published by Government of Tamilnadu, 1979.

2.     Thiyagarajan. R. Text Book of Materia Medica (Gunapadam)- Thathu and Jeevam, Department of Indian Medicine and Homoeopathy, 2008.

3.     Kannusaami Pillai C, Sikicha Rathina Deepam ennum Vaithiya nool, B.Rathina Naicker and Sons, Chennai- 600049, 2007.

4.     Sarakku Suthi Sei Muraigal, Tamil Nool varisai-8, Thamarai Noolagam, Chennai.

5.     Lohar DR. Protocol for testing: Ayurvedic, Siddha and Unani Medicines Pharmacopoeial Laboratory for Indian Medicine, Ghaziabad. Mumbai, revised edition.

6.     OECD. Guidance Document on Acute Oral Toxicity. Environmental Health and Safety Monograph Series on Testing and Assessment No 24, 2000.

7.     OECD. Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation Environmental Health and Safety Monograph Series on Testing and Assessment No 19, 2000.

8.     RL Lipnick; JA Cotruvo; RN Hill; RD Bruce; KA Stitzel; AP Walker; I Chu; M Goddard; L Segal; JA Springer; RC Myers. Food Chem Toxicol. 1995, 33(3), 223-231.

9.     Soane  K.M. Chaves,  Chistiane  M. Feitosa and   Lidiane  da S. Araújo, Alkaloids Pharmacological Activities - Prospects for the Development of Phyto pharmaceuticals for Neurodegenerative Diseases, Current Pharmaceutical Biotechnology, volume 17, issue 7, pages 629-635, year 2016, issn 1389-2010/1873-4316, (http://www.eurekaselect.com/node/141720/article).

10.   Kumar, Rakesh and Anjum, Nishat and Tripathi, Yogesh. (2015). Phytochemistry and Pharmacology of Santalum album L.: A Review. World Journal of Pharmaceutical Research. 4. 1842-1876.

 

 

 

 

 

 

Accepted on 21.11.2018       © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(1): 227-230.