Deshpande Shailesh V, Rashmi Chojer* and Deshpande Vaishali S.

Department of Kayachikitsa, Parul Institute of Ayurved, Parul University, AP Limda, Tal – Waghodia, Vadodara, Gujarat, India.

Corresponding Author E-mail: rashmi.chojer@gmail.com

Article Publishing History
Article Received on : 19 Jul 2023
Article Accepted on :
Article Published : 17 Oct 2023

ABSTRACT:

Introduction: Kanakasava is the classical formulation known to have beneficial effects on bronchial asthma. This study deals with the objective to compare the phytochemicals present in Kanakasava, Kanakasava distillate, and Kanakasava nebulizer solution using gas chromatography-mass spectrometry (GC-MS) and to determine if Kanakasava nebulizer solution can be useful in managing respiratory diseases. Methods: Kanakasava, Kanakasava distillate and Kanakasava nebulizer solution were prepared accordingly and standard GC-MS analysis was performed on the samples. Results: The GC-MS analysis indicated the presence of 6, 13 and 10 important phytochemicals in Kanakasava, Kanakasava distillate and Kanakasava nebulizer solution respectively. Many chemicals acting on the respiratory system were found in all three forms of Kanakasava and some of the chemicals were having similar actions. Conclusion: Despite the fact that the phytochemicals found in the three of them differed due to molecular fragmentation during the whole process, the efficacy of the novel Kanakasava nebulizer solution remained unaffected.

KEYWORDS:

Ayurveda; GC-MS; Herbal nebulizer solution; Kanakasava; Molecular fragmentation

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Shailesh D, Chojer R, Vaishali S. D. Comparative Phytochemical Analysis of Kanakasava, Kanakasava Distillate, and Kanakasava Nebulizer Solution Using Gas Chromatography-Mass Spectrometry (GC-MS). Orient J Chem 2023;39(5).

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Shailesh D, Chojer R, Vaishali S. D. Comparative Phytochemical Analysis of Kanakasava, Kanakasava Distillate, and Kanakasava Nebulizer Solution Using Gas Chromatography-Mass Spectrometry (GC-MS). Orient J Chem 2023;39(5). Available from: https://bit.ly/3PY3qCu

Introduction

Chronic respiratory disorders, which can affect both adults and children, are becoming more and more prevalent worldwide. According to WHO, an estimated 400 million individuals worldwide suffer from asthma and chronic obstructive pulmonary disease (COPD) alone 1. COPD prevalence is anticipated to increase over the next 40 years, with over 5.4 million deaths from COPD and related illnesses occurring annually by 2060 2.Despite a constant increase in the burden of chronic respiratory diseases, modern treatment including inhaled and oral corticosteroids, long and short-acting bronchodilators is not adequate and safe enough to manage respiratory diseases like asthma completely.

Many side effects like migraine, dry mouth, and tachycardia are seen with the prolonged use of modern anti-asthmatic therapy and studies have also shown that up to 80% of asthma-related deaths are caused by common asthma inhalers 3. Considering the side effects that occur during modern therapy, there is a need to explore the traditional system of medicines that provides long-lasting and safe management for asthma4.

Kanakasava is a classical Ayurvedic polyherbal formulation in which individual drug consists of some chemical constituents that are known to have anti-asthmatic, anti-allergy, anti-tussive, and bronchodilator actions. It makes the Kanakasava, a potent anti-asthmatic formulation when taken orally. It is a self-fermented formulation (Sandhana) and is useful in asthma (shwasa), cough (kasa), yakshma (tuberculosis), kshatksheena (~phthisis), chronic fever (jeerna jwara), and raktapitta (~bleeding disorders)5. The immunostimulating activity of Kanakasava is proven through an in-vitro study which is seen due to its capacity to increase antibody production and splenocyte proliferation6. An animal study found Kanakasava to be effective against ovalbumin-induced bronchial asthma and airway inflammation in rats 7. The clinical efficacy of Kanakasava is also seen in the management of bronchial asthma as it provides symptomatic relief in the patients and statistically significant changes are seen in PEFR and FEV1 values8. Therefore, it can be said that Kanakasava can be effective in the management of chronic respiratory diseases like asthma, COPD.

In cases of COPD or other diseases of the respiratory system, a medicine that can be directly administered into the lungs can show better and faster effects as compared to other routes. So, drug delivery through nebulizers or rota healers is highly effective. Hence, an effort was made to convert the Kanakasava into a nebulizer solution and to evaluate its efficacy in the management of respiratory diseases.

The current study was carried out with the goal of comparing the phytochemicals present in Kanakasava, Kanakasava distillate, and Kanakasava nebulizer solution using gas chromatography-mass spectrometry (GC-MS) and to evaluate whether the Kanakasava nebulizer solution has the potential to manage chronic respiratory diseases or not.

Materials and Methods

Collection of Raw Plant Material

All the crude drugs were collected and authenticated by an in-house expert botanist. The details of the ingredients are outlined in Table 1

Table 1: Kanakasava Composition

S.NO.

Name of the Drug

Botanical Name

Part used

Chemical Constituent

Action

1.      

Kanaka

Datura Stramonium Linn

Whole plant

Atropine, Scopolamine

Anticholinergic,9 Bronchodilator9

2.      

Vasa

Adhatoda vasica Nees

Whole plant

Vasicine (Bromhexine and Ambroxol), Vasicinone, Vascinol

Anti-asthmatic,10 Bronchodilator,11 Anti-allergy,11 Anti tubercular11

3.      

Madhuka

Glycyrrhiza glabra Linn.

Root

Glycyrrhizin, Liquiritin, Liquiritigenin

Anti-tussive,12 Expectorant12

4.      

Pippali

Piper longum Linn

Fruits

Piperine, Piplartine

Anti asthmatic,13 Bronchodilator,13 Anti inflammatory13

5.      

Kantakari

Solanum xanthocarpum Scrad & Wendl

Whole plant

Solanacarpine, Solamargine, Solasodine

Anti inflammatory,14 Anti- asthmatic,14 Anti-tussive

6.      

Nagakesar

Mesua ferrea Linn

Stamen

Mesuaxanthone A, Mesuaxanthone B

Anti inflammatory15

7.      

Shunthi

Zingiber officinalis Rosc

Rhizome

Zingerone, Gingerol

Anti inflammatory16

8.      

Bharangi

Clerodendrum serratum (Linn.)

Root

Catechin, Luteolin

Anti asthmatic,17 Anti histaminic,17 Anti-allergy17

9.      

Talispatra

Abies webbiana Lindl

Leaves

Saponins (Icosahydropicenic acid)

Anti-inflammatory18

10.   

Dhataki

Woodfordia fruticosa (L.) Kurz.

Flowers

Octasonal, diglucoside, and beta-sitosterol.

Relieves cough19

 

Preparation of Kanakasava

Kanakasava was prepared according to the classical decoction method mentioned in Bhaishajya Ratnavali.20

All the crude drugs were initially cleaned, shade dried, powdered, and sieved. The powdered drugs were soaked in 25 litres of water and were kept undisturbed for 24 hours. Then, a porcelain jar was selected and Dhoopana was done using the Dhoopana Dravyas (Guggulu, Tulsi, Neem). Later the mixture was poured into the jar and at the end, Draksha (Vitis Vinifera) and flowers of Dhataki (Woodfordia fruticosa) were added and the container was sealed with a clay-smeared cloth. The container was kept undisturbed in a dark room for 38 days till the completion of fermentation. The following observations were used to confirm fermentation:

Asava prepared possessed an alcoholic odour.

No evidence of effervescence was seen.

Burning candle test was positive.

After the confirmation of fermentation, the prepared formulation i.e., Kanakasava (4 litres) was filtered through a double-layered clean cotton cloth, stored in a glass flask and was subjected to distillation.

Preparation of Kanakasava Distillate

The distillate of Kanakasava was obtained through a simple distillation method.21 The following procedure was used to obtain the distillate:

The distillation apparatus was set up which included a distillation flask, a condenser, and a receiving flask. The Kanakasava was poured into the distillation flask and was heated at 40 degree Celsius (0C)  until it started boiling. As the Asava boils, the vapor rises and enters the condenser. The condenser cools the vapor, causing it to condense back into a liquid, which was collected in the receiving flask. The liquid collected in the receiving flask is the Kanakasava distillate (2 litres).

Preparation of Kanakasava Nebulizer Solution

The distillate thus obtained was diluted with distilled water in a ratio of 2:1, which was finalized after various trials as per the concentration suitable for nasal mucosa. The final solution obtained was the Kanakasava nebulizer solution which can be used directly for the nebulization.

Gas Chromatography-Mass Spectrometry Analysis

 The GC-MS study was done by the protocols already available in the literature.22

The sample medicine was analysed using a GC-MS Perkin Elmer System, which included an autosampler and a gas chromatograph interfaced to a mass spectrometer (GCMS) apparatus, under the following conditions:

The column used was Elite-5MS (30 meters*0.250mm*0.250um). Helium was used as a carrier gas at a constant flow rate of 1ml/min, with an injection volume of 2 microlitres and an injector temperature of 260°C.The oven temperature was programmed to rise from 75°C (isothermal for 5 minutes) to 280°C at a rate of 10°C/min, then fall for 10 minutes at 280°C.

The temperature of the EI source was 220 degree Celsius. At a scan range of 20 to 610 amu, mass spectra were collected. The total running time for the GC was 45 minutes.

Identification of Compounds

For mass spectrum GC-MS interpretation, the National Institute of Standards and Technology (NIST) online database was used. The unknown component’s mass spectrum was compared to the spectrum of known components listed in the NIST online database.22

Results

The phytochemicals found in Kanakasava, Kanakasava Distillate, and Kanakasava nebulizer Solution were compared.  They are listed below in Table 2, Table 3, and Table 4 respectively. [*Source: PubChem and Dr. Duke’s phytochemical and ethnobotanical database (online database)]

Table 2: Phytochemicals found in Kanakasava

S.NO

Name of Compound

Molecular Name

Molecular weight (g/mol)

Activity*

1

Ethyl 4-T-Butylbenzoate

C13H18O2

206

Anti-microbial, Metabolite, Anti-tumor, Blood thinning

2

Pentanedioic acid

C19H28O4

320

Metabolite, Acidifier, Anti-inflammatory, Anti-allergy

3

Phenol,2,5-BIS (1,1-Dimethylethyl)

C14H22O

206

Anti-oxidant, Anti-microbial, Anti-fungal, Anti-inflammatory,23 Anti tuberculotic activity

4

Undecanoic acid

C13H26O2

214

Antioxidant24, inhibit production of Uric Acid, Anti fungal25

5

Decanoic acid

C12H24O2

200

Anti-bacterial26, Anti-fungal,26 Anti-inflammatory, Metabolite

6

Ethyl Tridecanoate

C15H30O2

242

Anti-microbial27, Anti-inflammatory27

Table 3: Phytochemicals found in Kanakasava distillate

S.NO

Name of Compound

Molecular Name

Molecular Weight

(g/mol)

Activity*

1

Malonic Acid, Dihydroxy-diisobutyl ester

C11H20O6

248

Controlling acidity, Metabolite, Anti-inflammatory28, Anti -bacterial action

2

Propane, 2-(1,1-Dimethylethyl Sulfonyl)-2-Methyl

C8H18O2S

178

Anti-bacterial, Anti-fungal,29 Anti oxidant29

3

Oxalic acid, Bis(isobutyl) ester

C10H18O4

202

Antioxidant, Antimicrobial

4

Triarachine

C63H122O6

974

Anti-Microbial

5

Dodecyl Nonyl ether

C19H3202S

324

Anti-Microbial

6

Heptyl Hexadecyl ether

C26H54

366

Anti-Microbial

7

Lauroyl Peroxide

C24H46O4

398

 

 

 

Anti -helminthic30, Anti-protozoal30, Anti-viral30, Anti-fungal30

8

Heptyl Octacosyl ether

C35H720

508

Antimicrobial31, Anti-bacterial, Antioxidant31

9

Sulfurous acid, Cyclohexylmethyl Hexadecyl ester

C23H46O3S

402

Anti-tumor32, Antibacterial32, Anti-Cancer33

10

Pimelic acid

C26H50O4

426

E coli Metabolite

11

Trimethylpentyl Undecyl ester

C26H50O4

426

Anti-microbial

12

Diethylmalonic acid

C23H44O4

384

Synthesis of Anti-inflammatory agents, Flavoring agents, Controls acidity

13

 Propanoic acid, 3,3’-thiobis-didodecyl ester

C30H580O4S

514

Anti-microbial, Anti-leukotrienic agent34

 

Table 4: Phytochemicals found in Kanakasava nebulizer solution

S.NO.

Name of Compound

Molecular Name

Molecular Weight

(g/mol)

Activity*

1

Salicyl Hydrazide

C7H8O2N2

152

Anti-microbial35, Anti-bacterial, Anti-fungal

2

Phenyl Salicylate

C13H10O3

214

Anti-bacteria36, Anti-inflammatory

3

3-Eicosene

C20H40

280

Anti-microbial37, Antioxidant38, Anti-inflammatory39

4

Cetene

C16H32

224

Anti-microbial39, Anti-oxidant39

5

Trichloroacetic acid

C18H33O2C13

386

Metabolite, used for treating acne, warts

6

Tetracosanol-1

C24H50O

354

Anti-Mutagenic40, Antiseptic, Anti-inflammatory41, Anti -bacterial42

7

Heptacosanol

C27H56O

396

(Flavouring agent, cholesterol Lowering, Antimicrobial and Antithrombotic)42

8

Cyclooctacosane

C28H56

392

Anti-fungal, Anti-bacterial, Anti-inflammatory

9

Tricosene

C23H46

322

Anti-bacterial43

10

Pentadecafluorooctanoic acid, Pentadecyl ester

C23H31O2F15

624

Anti-inflammatory43

 

Phytochemicals observed in all the three forms of Kanakasava, possess similar action on respiratory system. Details of the same are compiled in tables 5 and 6.

Table 5: Chemical compounds with actions on the respiratory system

S.NO

ACTION

KANAKASAVA

KANAKASAVA DISTILLATE

KANAKASAVA NEBULIZER SOLUTION

1.

Anti-inflammatory

Pentanedioic acid, Phenol,2,5-BIS (1,1-Dimethylethyl), Decanoic Acid, Ethyl Tridecanoate

Malonic Acid, Dihydroxy-diisobutyl ester, Diethylmalonic Acid

Phenyl Salicylate, 3-Eicosene, Tetracosanol-1, Cyclooctacosane, Pentadecafluorooctanoic Acid, Pentadecyl Ester

2.

Anti-bacterial

Decanoic Acid

Malonic Acid, Dihydroxy-diisobutyl ester, Propane, 2-(1,1-Dimethylethyl Sulfonyl)-2-Methyl, Heptyl Octacosyl Ether, Sulfurous acid, Cyclohexylmethyl Hexadecyl Ester

Salicyl Hydrazide, Phenyl Salicylate, Tetracosanol-1, Cyclooctacosane, Tricosene

3.

Anti-fungal

Phenol,2,5-BIS (1,1-Dimethylethyl), Undecanoic Acid, Decanoic Acid

Propane, 2-(1,1-Dimethylethyl Sulfonyl)-2-Methyl, Lauroyl Peroxide,

Salicyl Hydrazide, Cyclooctacosane

4.

Anti-microbial

Ethyl 4-T-Butylbenzoate, Phenol,2,5-BIS (1,1-Dimethylethyl), Ethyl Tridecanoate

Oxalic Acid, Bis(isobutyl) Ester, Triarachine, Dodecyl Nonyl Ether, Heptyl Hexadecyl Ether, Heptyl Octacosyl Ether, Trimethylpentyl Undecyl Ester, Propanoic acid, 3,3’-thiobis-didodecyl Ester

Salicyl Hydrazide, 3-Eicosene, Cetene, Heptacosanol

5.

Anti-oxidant

Phenol,2,5-BIS (1,1-Dimethylethyl), Undecanoic Acid

Propane, 2-(1,1-Dimethylethyl Sulfonyl)-2-Methyl, Oxalic Acid, Bis(isobutyl) Ester, Heptyl Octacosyl Ether

3-Eicosene, Cetene

6.

Anti-tuberculotic activity

Phenol,2,5-BIS (1,1-Dimethylethyl)

 

 

7.

Anti-leukotrienic agent

 

Propanoic acid, 3,3’-thiobis-didodecyl Ester

 

 

Table 6: Chemical compounds having similar actions

 Chemical compounds

Sample

Action

Decanoic acid44

Kanakasava

Anti-bacterial action against Staphylococcus Aureus

Sulfurous acid, Cyclohexylmethyl Hexadecyl Ester45

Kanakasava Distillate

3-Eicosene46, Phenyl Salicylate47

Kanakasava Nebulizer Solution

 

Discussion

This study shows that although molecule fragmentation has increased from fermented form (Asava) to nebulizer form, many of the molecular fragments are dissociated into a composite chemical form. It is seen that through the fragmentation of molecules, new chemicals have been identified which has been the case with Kanakasava nebulizer. In Kanakasava, a total of 6 identifiable compounds were found, in Kanakasava distillate, 13 identifiable compounds were found and in Kanakasava nebulizer solution, 10 identifiable compounds were found.

Factors like high temperature, type of water (distilled, deionized, or tap water) used for the dilution, atmospheric conditions, or variation in the pH of the solutions after the distillation or dilution process can affect the fragmentation of molecules in any experiment. However, in this study, high temperature, atmospheric conditions or variations in the pH of the solutions after the distillation or dilution process might have contributed to the fragmentation of molecules.

Even though there were a number of steps involved in the process and it is possible that some sensitive organic molecules might have resulted in fragmentation during different steps under GC-MS and our results indicate that fragmentation at different steps does not affect the effectiveness of the novel nebulizer solution reported in this work.

The molecular fragments obtained are of the finest forms of organic compounds and they show similar actions like antibacterial action, anti-inflammatory action which were found in all three forms of Kanakasava (as shown in Table no 4.)

For example, all forms of Kanakasava, including Kanakasava, Kanakasava distillate, and Kanakasava nebulizer solution, have shown anti-bacterial activity against Staphylococcus aureus which exerts a pathogenetic role in many chronic airway illnesses, such as COPD, asthma, pneumonia, etc.[48] (Table no.5)

This indicates that both the Kanakasava and the Kanakasava nebulizer solution would aid in preventing and treating chronic respiratory infections. With the help of the finest fragments present in the Kanakasava nebulizer solution, respiratory disorders can be treated more successfully by ensuring the targeted delivery of the necessary medication.

Limitations and Future Scope of the Study

To identify more phytochemical elements in detail in all three forms, various analytical tests, such as LCMS and HPTLC, can be conducted. The results of all the analytical tests can then be compared. Kanakasava nebulizer solution can go through all four stages of clinical trials following a thorough examination of the drug so that it can be utilized in the future to treat chronic respiratory disorders like COPD and asthma.

Conclusion

When a fundamental formulation is transformed into various new forms, the phytochemicals that result may vary due to various preparation methods and proper molecular fragmentation, but the new forms will still exhibit the same action as the fundamental formulation, only the chemical composition of molecules may change. Additionally, converting the well-known asthma medication Kanakasava into a nebulizer solution can aid in the treatment of respiratory illnesses.

Acknowledgement

Authors duly acknowledge SICART [Sophisticated Instrumentation Centre for Applied Research Testing], Anand, Gujarat for conducting GC-MS Analysis of all three formulations. We also thank Dr Rajat Arora, PhD Biochemistry for her valuable guidance.

Conflict of Interest

There is no conflict of interest, according to the authors.

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