Comparative Study of Different Samples of Chandamarutha sindoora for Neuro-Regenerative Activity – An Experimental Study

Ayurveda has a long tradition of utilizing Herbo-mineral formulations for the management of chronic and difficult-to-treat disorders. These formulations (Rasaushadhis) occupy a significant place in Ayurvedic therapeutics due to their rapid action, smaller dose, enhanced bioavailability, and long shelf life. The components of these formulations undergo specific Shodhana (purification), Marana (Incineration) etc pharmaceutical processing, which are believed to enhance their therapeutic efficacy while minimizing their toxicities.

Among these formulations, Chandamarutha Sindoora is a well-known herbo-mineral preparation mentioned in both Ayurvedic and Siddha system, containing purified mercurial compounds and Sulphur as major ingredients. However, variations in ingredient proportions and manufacturing methods may influence its physicochemical characteristics, safety, and therapeutic efficacy.

The Chandamarutha Sindoora (CMS) is described in several classical texts including Rasayoga Sagara, Agastya Vaidya Chandrika and Ayurveda Vijnana Kosham where it is primarily indicated for Neurological conditions such as Pakshaghata. In the present study three different samples of Chandamarutha Sindoora were prepared based on the formulations described in these texts. The Chandamarutha Sindoora containing ingredients such as Rasa Sindoora, Rasa Karpoora, Rasa Pushpa, Hingula and Gandhaka, combined in varying proportions, according to the respective textual references and compared to identify the most efficacious formulation with superior Neuro-regenerative and neuroprotective activity.

Among Siddha formulations, Chandamarutha Chendooram has been traditionally indicated for conditions such as paralysis, facial palsy, hemiplegia, neuromuscular disorders, and other degenerative diseases.

Pakshaghata, described under Vata Vyadhi in Ayurveda, is characterized by loss of motor function, weakness, pain, and impaired movements affecting one half of the body. Clinically, it closely resembles Hemiplegia, which commonly occurs following cerebrovascular accidents (stroke).¹ Stroke is one of the leading causes of long-term disability worldwide, resulting in significant physical, psychological, social, and economic burden.² Although the available stroke management by different medical systems mainly focuses on thrombolysis, rehabilitation, and symptomatic care.³ Complete neurological recovery remains challenging, necessitating the exploration of effective complementary therapeutic approaches.

Neurodegeneration involves oxidative stress, neuroinflammation, excitotoxicity, and progressive neuronal loss. Hence, there is growing interest in scientifically validating traditional formulations that may possess neuroprotective properties.⁴

Recent experimental studies have demonstrated that Chandamarutha Chendooram exhibits significant neuroprotective activity by reducing oxidative stress, neuroinflammation, and neuronal damage in experimental models. These findings support the need for further pharmaceutical processing and experimental validation of the formulation.⁵

Ayurvedic classics advocate the use of Vatahara, Balya, Rasayana, and Medhya drugs in the management of Pakshaghata. Balya, and neuroprotective properties may help to improve nerve conduction, reduce neuronal damage, promote tissue regeneration, and enhance functional recovery.

Therefore, the present study is undertaken to prepare Chandamarutha Sindoora according to classical methods, evaluate its pharmaceutical and analytical characteristics, and experimentally assess its neuroprotective efficacy, in Aluminium Chloride induced Neuro-degeneration model in Wistar Albino rats. The study aimed to generate scientific evidence supporting safety, quality and therapeutic potential of this classical formulation.

MATERIALS AND METHODS

Pharmaceutical study:

1. Identification, collection and purification of raw drugs:

The ingredients required for the preparation of Chandamarutha Sindoora are collected from local market and are analysed for genuinity. The Chandamarutha Sindhoora contains intermediate products like Rasa Karpoora (pooram), Rasa Pushpa (veeram), Hingula and Gandhaka, which are subjected to purification process. The Rasa Sindoora purchased from the market was added during the procedure without purification. Rasa Karpoora and Rasa Pushpa Shodhana was done according to Siddha source of literature. Hingula and Gandhaka Shodhana was done according to Rasa Tarangini and Ayurveda Prakasha text books.

2. Preparation of Chandamarutha Sindoora:

All the ingredients were taken, weighed according to the different ratios as mentioned in the texts such as Rasayoga Sagara, Agastya Vaidya Chandrika and Ayurveda Vijnana Kosham and were triturated with egg white until it became red coloured Sindoora form. This methodology was explained under the category of Chendooram preparation in Siddha medicine.

1. Veeram Shodhana or Rasa Pushpa shodhana:

It was performed according to Siddha Formulary of India.

• 20g of Camphor was taken, grinded to powder and mixed with 800ml of Tender Coconut water. It was taken in a mud pot.
• Pottali of Veeram or Rasa Pushpa was made with a clean Kora cloth and was hung from a wooden stick, which was kept over the mud pot to form a Dola Yantra.
• Pottali height was adjusted so as to stay it above the liquid throughout the procedure.
• Steaming of the Pottali was done for 1 hour after which the purified Veeram or Rasa Pushpa was collected dried under the Sun and stored in a glass container.

2. Pooram Shodhana (Rasa Karpoora) Shodhana:

It was performed according to Siddha Formulary of India.

• Nagavalli Patra was washed with water to remove impurities and ground with Maricha to form a Kalka in a clean and dry Khalwa Yantra.
• The above prepared Kalka was added to 1350 ml of water in a mud pot. Pottali of Rasa Karpoora was made with a clean Cora cloth and tied to a wooden stick.
• The Pottali was suspended in the above liquid till it gets completely immersed and the pot was kept over the gas stove for heating.
• Continued the heating till the liquid was reduced to 1/4^th portion.
• When cooled the Pottali was removed, Rasa Karpoora was collected and washed with warm water. Then it was dried and stored in a glass Jar.

3. Gandhaka Shodhana⁶

It was performed according to A.Ρ. 2/21-24

• Gandhaka was grinded to powder form.
• In an Iron pan equal amount of Ghee was taken and melted over Mandagni.
• To this powdered Ashuddha Gandhaka was added. when all the Gandhaka got melted, it was filtered through the cloth into a vessel containing milk.
• Milk was then discarded & Gandhaka was washed thoroughly with hot water and then kept for drying under shade.
• After drying, it was powdered again and the same procedure was repeated for 2 more times.
• After completion of the process, Shuddha Gandhaka was dried, powdered and stored in a glass jar.

4. Hingula Shodhana⁷

It was performed according to Rasa Tarangini 9/12

• Ashuddha Hingula was taken and powdered in a Khalwa Yantra.
• Ardraka Swarasa was added to it and Bhavana was carried out till the Hingula got completely dried.
• This was repeated for 7 times by adding fresh Ardraka Swarasa during each Bhavana.
• After 7^th Bhavana the Shodhita Hingula was collected and dried under shade.

5. CHANDAMARUTHA SINDOORA  (CMS) - 1

Reference: AYURVEDA VIJNANA KOSHAM ⁸

Procedure

• Each ingredient was taken according to the ratio mentioned in the text and triturated separately to made into fine powder.
• Then it was added one by one in to a Khalwa Yantra and triturated in clockwise direction with a stroke of 25/min.
• 24ml of Egg white was added and Bhavana was done until the whole Egg white was dried and dry powder of Sindoora coloured preparation was obtained.

6. CHANDAMARUTHA SINDOORA (CMS) - 2

Reference: RASAYOGA SAGARA⁹

Ingredients and Quantity:

Procedure

• Each ingredient was triturated separately and made into fine powder. Then the powder was added one by one in to a Khalwa Yantra and trituration was continued at the rate of 25strokes/min to made into a homogenous mixture.
• Egg white was added to the mixture after filtering and Bhavana was done until the whole Egg white was dried and Sindoora like preparation was obtained. It took around 6hrs for completing the procedure.

7. CHANDAMARUTHA SINDOORA (CMS) - 3

Reference:  AGASTYA VAIDYA CHANDRIKA¹⁰

Ingredient and Quantity:

Procedure:

• Each ingredient was triturated and made into fine powder separately.
• Then the ingredients were added to a Khalwa yantra and sufficient quantity of Egg white was added and Bhavana was done until the whole Egg white was dried and Sindoora like preparation was obtained.
• It took around 6hrs for completing the procedure.

Experimental study:

Wistar albino rats were taken randomly from the well- established animal house attached to S.D.M. Centre for Research in Ayurveda and Allied Sciences.

An experimental model for the study of Neuroregenerative activity is selected in such way that it would satisfy the following condition:

• To evaluate the Neuroregenerative action of Chandamarutha Sindoora on Aluminium chloride-induced Neurotoxicity.
• To estimate Neuroregenerative activity of 3 samples of Chandamarutha Sindoora and to determine the most efficacious sample.

Table no. 2.2 showing grouping of rats

Group 1: The rats were given normal tap water & Standard laboratory diet.

Group 2: The rats were injected with Aluminium Chloride for 45 days.

Group 3: The rats were injected with Aluminium Chloride (AlCl₃) followed by trial drug Chandamarutha Sindoora -1, administered orally through feeding tube after 1 hour of injection for 45 days.

Group 4: The rats were injected with Aluminium Chloride (AlCl₃) followed by trial drug Chandamarutha Sindoora - 2 administered orally through feeding tube after 1 hour of injection for 45 days.

Group 5: The rats were injected with Aluminium Chloride (AlCl₃) followed by trial drug Chandamarutha Sindoora - 3 administered orally through feeding tube after 1 hour of injection for 45 days.

Body weight, open field behaviour test and behavioural despair test was conducted on 7th day from the day of administration of dosing. On 45th day the same tests were conducted and the parameters such as number of outer squares, middle squares, inner squares crossed, rearing, grooming, faecal pellets and freezing time was assessed in every rat of each group and values were noted.

From 15th day onwards training for Cook’s Pole Climbing apparatus test was started for 7 days and on 7th day assessment of retention of Conditioned Avoidance Response was conducted in each rat in each group and the values were noted.

On 45th day all rats from each group were Anaesthetised and blood collected from the Orbital Plexus for Blood Acetyl-cholinesterase test and hours later 2 rats from each group were sacrificed through deep ether Anaesthesia and Brain was removed by dissection and weighed and sent for Histopathology studies by storing in 10% Formalin.

RESULTS:

Results of pharmaceutical study 

Table no.3.1 showing results of Shodhana of ingredients.

Table no 3.2 showing final result of Chandamarutha Sindoora

Results of analytical study

Organoleptic characters were analysed. Sample 1 and 2 were bright red in colour and sample 3 was dark red in colour with faint characteristic odour.  All the samples were tasteless and lustreless.

    

Fig 1 Raw Rasa karpoora                      Fig 2 Rasa karpoora shodhana

    

Fig 3 Raw Rasa Pushpa                         Fig 4 Rasa Pushpa Shodhana

    Fig 5 Melted Gandhaka                         Fig 6 Melted Gandhaka in Ksheera

    

Fig 7 Shodhita hingula                           Fig 8 Rasa Sindoora

    

Fig 9 Preparing CMS                                Fig 10 Chandamarutha sindoora

    

Fig 11 Injecting AlCl3                                 Fig 12 Behavioural despair test

    

Fig 13 Open field test                                  Fig 14 Cook’s pole climbing test

CONCLUSION:

• According to the conceptual study, the drug CMS possesses Prabhava due to its unique combination of ingredients during pharmaceutical processing’s. Prabhava remains the highest in the hierarchy of pharmacological actions of a drug especially the Rasoushadhi’s. As Rasa Dravyas are useful in Asadhya Rogas, Chandamarutha Sindoora has the potential to treat many severe diseases including Pakshaghata, Vatavyadhi, Arbuda, Granthi, and Mahodara etc.
• Pharmaceutically, Chandamarutha Sindoora can be prepared with different proportions of Rasa Karpoora, Rasa Pushpa, Gandhaka, Hingula, and Rasa Sindoora. The classical method of processing is uncommon and should be carried out with utmost care. The formulation can be prepared by following the procedure adopted in the present study. To make the pharmaceutical process easier, Rasa Karpura and Rasa Pushpa were purchased from the market and relevant Shodhana techniques were carried out as mentioned in the Siddha practices. Rasa Sindoora was bought from the local market, Hingula and Gandhaka were purified according to Ayurvedic classics before mixing them in different ratios and Bhavana was given with Egg white.
• Experimental evaluation in the AlCl₃ - induced Neurodegenerative model of Rats demonstrated improvement in Open field test Behavioural parameters, Cholinesterase activity, Brain weight and Histopathological changes in Brain tissue. Among the three formulations of Chendamarutha Sindoora samples, CMS 3 exhibited comparatively better neuroprotective and Neuroregenerative activity followed by CMS 2 and CMS 1.
• Based on the findings of the present study, it can be concluded that Chandamarutha Sindoora prepared according to the classical references possesses significant potential in managing Neurodegenerative conditions associated with neuronal damage.

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