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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 6
| Issue : 1 | Page : 26-35 |
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Drug standardization and in vitro wound healing potential of Siddha herbomineral formulation Kandhaga Ennai for diabetic ulcers
Kehren R Rayma, Kirutisri Karthigeyan, Pooja Muniraj, Jeeva Gladys, Balagurusamy Karthigaeyam
Velumailu Siddha Medical College and Hospital, Sriperumbudur, Tamil Nadu, India
| Date of Submission | 11-May-2023 |
| Date of Decision | 12-Jul-2023 |
| Date of Acceptance | 13-Jul-2023 |
| Date of Web Publication | 29-Aug-2023 |
Correspondence Address:
Kehren R Rayma
Velumailu Siddha Medical College and Hospital, Kancheepuram, Sriperumbudur – 602 105, Tamil Nadu
India
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/jrsm.jrsm_7_23
Background: A serious and often expensive consequence of diabetes mellitus and diabetic foot ulcers are a major global source of death and morbidity. A diabetic patient’s lifetime risk of developing a foot ulcer has been estimated to be between 19 and 34%. Objective: The study drug Kandhaga Ennai (KE) selected from the Siddha classical text Rasavatha Manjari by P.M. Abdullah Saayabu is taken for preliminary assessing its wound healing potential of Ranamegangal (diabetic ulcers). Materials and Methods: The drug is prepared as per the method mentioned in the classic Siddha literature. KE is subjected to a sophisticated instrumental analysis such as Fourier transform infrared (FTIR) inductively coupled plasma optical emission spectrometry studies, a few physicochemical analytical parameters, and in vitro preclinical studies toward wound healing assays. Results: The study infers that all the toxic heavy metals are within range. KE has significant antioxidant activities reducing free radicals and effective antifungal property, and promotes migration and proliferation of fibroblasts. The results indicate that the herbomineral formulation may be useful in effective management of superficial wounds. Conclusion: This study validates the traditional claim of KE confirming the herbomineral formulation for anti-inflammatory, antioxidant, antimicrobial, and wound healing activities through in vitro methods. Additional experimental animal tests as well as clinical trials are required of this drug to treat diabetic ulcers. Keywords: Antibacterial, antifungal, anti, inflammatory, antioxidant, Siddha
How to cite this article:
Rayma KR, Karthigeyan K, Muniraj P, Gladys J, Karthigaeyam B. Drug standardization and in vitro wound healing potential of Siddha herbomineral formulation Kandhaga Ennai for diabetic ulcers. J Res Siddha Med 2023;6:26-35 |
How to cite this URL:
Rayma KR, Karthigeyan K, Muniraj P, Gladys J, Karthigaeyam B. Drug standardization and in vitro wound healing potential of Siddha herbomineral formulation Kandhaga Ennai for diabetic ulcers. J Res Siddha Med [serial online] 2023 [cited 2023 Oct 3];6:26-35. Available from: http://www.jrsm.in/text.asp?2023/6/1/26/384580 |
| Introduction | |  |
The International Diabetes Federation evaluates the complete number of individuals in India with diabetes, and estimated that the rates are ascending and may reach a peak of 783.2 million by 2045.[1] It is one of the leading causes of nontraumatic amputations of the lower extremities in developed countries and approximately 24% of diabetic patients with foot ulcer undergo amputation.[2] Upon injury to the skin, a set of complex biochemical events takes place in a closely orchestrated cascade to repair the damage. The classic model of wound healing comprises four sequential overlapping phases such as hemostasis, inflammation, proliferation, and remodeling. Currently, available treatments are limited due to side effects and cost-effectiveness. Hence, there exists a demand of exploring alternative, novel, efficacious, safer, and less expensive antidiabetic and wound healing agents as a stand-alone or combinatorial therapy.[3]Kandhaga Ennai (KE), a Siddha classical herbomineral formulation, is unique due to its claim in its effectiveness against “Ranamegangal” (diabetic ulcers) even at a mild dosage of 1–3 drops. The ingredients “Kandhagam, Vetrilai, and KaduguEnnai” also have a scientific background of antidiabetic, antimicrobial, antioxidative, and anti-inflammatory properties.[4],[5],[6],[7] The results of the studies substantiate the traditional claims of KE and favor the Siddha system of medicine as evidence-based.
| Materials and methods | | |
KE is a Siddha classical herbomineral formulation consisting of three main ingredients enlisted in [Table 1].
Purification of “Kandhagam” (sulfur)
Leaves of “Maruthondri” (Lawsonia inermis) were grinded in a stone mortar and mixed with Cow’s curd. The mixture was kept in a mud pot and covered with a cotton cloth, and raw “Kandhagam” was placed over the cloth. The pot was closed with a lid and covered with a cloth dipped in mud paste and sealed completely. The pot was buried in the earth up to the mouth level, and cow dung cakes were arranged over the mouth of the pot and were set on fire. The “Kandhagam” (sulfur) kept above the cloth melted due to heat and got collected at the bottom of the pot. The processed “Kandhagam” (sulfur) was taken out, and this process was repeated for seven times with fresh mixture each time. [Figure 1] shows a photograph of the Kandhagam that has been purified seven times.
Procedure
Fresh leaves of “Vetrilai” (Piper betle) were used by removing the tip and stalk. Both “Kandhagam and Vetrilai” were vigorously grinded to obtain a paste consistency. It was further rolled like a thread over an iron rod with many rolls of cloth placed on top of it. This should be dipped in “Kadugu Ennai” (mustard oil) and burnt [Figure 2]. A ceramic vessel was placed below this to obtain the oil and stored it in a container [Figure 3]. If required, this process can be repeated again.[9]
Rationale behind selecting study drug
Previous studies revealing the individual pharmacological action of preparatory drugs used in KE are listed in [Table 2].
Siddha humoral concept on the pharmacokinetics of KE
The ingredient sulfur consists of the taste “Kaippu” (Ether + Space), which can treat psoriasis and smoke scaly appearance found in skin diseases. ‘Karppu’suvai (Ether + Fire) and heat property present in “Vetrilai and Kadugu Ennai” increases the action of KE. Based on Trihumoral theory, “Pitham” is located in the skin. Skin diseases and their inflammations are developed due to either increased “Vatham or Kabam.” Itching of the skin is caused by increase in “Kabam,” and the dry, scaly, and blackish discoloration is caused due to increased “Vatham.” Administration of KE internally may regulate the “Viyanan type of Vatham,” thereby treating the skin ailments due to its unctuous property [Figure 4].
| Results | | |
Physicochemical analysis
Herbomineral formulation of KE is subjected to various physicochemical parameters for standardization. The results are exhibited in [Table 3].
The color of the sample is black and the same in both the ordinary light and ultraviolet light, which proves that there are no radioactive compounds present in it.[9]
The pH of the drug KE occurs to be 6.9, which is slightly acidic. In oral administration, the acidic nature of the drug enhances rapid absorption in the stomach. The pH value influences the quality of the medicine. Under low pH value (presence of acidic substance), the contamination of herbomineral preparation is observed to be lower. This pH from minerals or phytochemicals was assessed to determine if sulfur should be included alone in the final product or whether the remaining components should also be included. The other remaining component was among the findings of this standardization investigation.[10]
Sophisticated instrumental analysis
FTIR analysis
The results of Fourier transform infrared (FTIR)-KE are discussed in the graph [Figure 5].
Absorption spectra produced by FTIR analysis give details on the chemical bonds and molecular structure of a substance. Identification of the lead molecule for additional study to clarify the structure of functional chemicals that are reasonable for their therapeutic efficacy requires characterization of herbal formulations. FTIR can distinguish between chemical bonds of organic materials, detect and identify organic contaminants, and identify water, phosphates, sulfates, nitrates, nitrites, and ammonium ions that are useful with solids, liquids, or gases. It does this by absorbing infrared radiation, which excites vibrational modes in the bond.
The absorbance peaks around 3415 cm−1 are corresponding to either for O-H of water/alcohol/carboxylic acid. The absorbance frequency around 2923 and 2853 cm−1 arises from the -C-H stretch of Aldehydes. Further, the bands around 1635–1245 cm−1 are corresponding to the bending frequency of alkenes, amide, aromatics, CH3 Bend, NO2, and C-O-C stretch.[9]
Inductively coupled plasma optical emission spectrometry
Equipment: simultaneous inductively coupled plasma-optical emission spectrometry, vista-PRO
Sample
A total of 0.5 g of KE (wt. 0.224570 g) is measured, and then resolved in a decomposition vessel with nitric acid into a 10 mL solution. The intensity of the emission is indicative of the concentration of the element within the sample. Partial spectral profile and analysis results are shown in [Table 4]. | Table 4: Partial spectral profile and analysis results of inductively coupled plasma optical emission spectrometry
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In the drug sample, the heavy toxic metals such as Hg, As, Cd, and Pb are in below detectable level, and this infers that they are not present. A total of 750 mg/dL sulfur was present, which is within the admissible level. The available calcium and phosphorous might be obtained from the herbals used in this formulation. This itself elaborates the intellectual approach in the use of herbs, metals, and minerals by the great Siddhars.[9]
Calcium is not only acting as a crucial clotting factor at the initial stages of the healing process, but it also plays an important role in the regulation of proliferation and differentiation of keratinocytes. Phosphate avoids or diminishes biological responses undesired in the treatment of chronic wounds that were stimulated by calcium, such as increased gelatinase activity or expression of inflammatory factors.[11]
In vitro preclinical studies of KE toward wound healing
1,1-diphenyl-2-picrylhydrazyl radical scavenging activity
The ability of dimethyl sulfoxide (DMSO) extract of KE to scavenge free radicals formed was assessed using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). In the extract, the maximum DPPH˙ radical scavenging activity was 34.5 ± 0.22 at 120 µg/mL concentration. DMSO extract of KE demonstrated high capacity for scavenging free radicals [Figure 6] and [Table 5] by reducing the stable DPPH radical to the pale pink color DDPH, and the reducing capacity increased with increasing concentration of the extract. The inhibitory concentration 50 (IC50) value in leaves was found to be 173.91 µg/mL concentration and was compared with standard (IC50 = 11.98 μg/mL concentration) ascorbic acid.[12]
Anti-inflammatory activity
Hemolysis is due to red blood cell destruction, which resulted from lysis of membrane lipid bilayer. Erythrocytes are considered a major target for the free radicals owing to the presence of both high membrane concentration of polyunsaturated fatty acids and the oxygen transport associated with redox-active hemoglobin molecules, which are potent promoters of activated oxygen species. The erythrocyte model has been widely used as the direct indication of toxicity of injectable formulations as well as a general indication of membrane toxicity.[13]
The DMSO extract of KE was to authenticate that traditional information by in vitro anti-inflammatory screening in the treatment of inflammation. The antidiabetic blood maximum hemolysis inhibition was 79.93 ± 0.22 at 120 µg/mL concentration [Table 6], and the IC50 was 45.02 μg/mL concentration. The hyperglycemic diabetic blood maximum hemolysis inhibition was 38.73 ± 0.22 at 120 µg/mL concentration [Table 7], and the IC50 was 154.91 μg/mL concentration. | Table 6: Anti-inflammatory activity of DMSO extract of KE in antidiabetic blood
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 | Table 7: Anti-inflammatory activity of DMSO extract of KE in diabetic blood
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Antibacterial activity by agar well diffusion method
The DMSO extract of KE was tested for its antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus, Bacillussubtilis, and Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. The antibacterial sensitivity of the extract and their potency were assessed quantitatively by measuring the diameter of the clear zone in cultures in Petri plates [Table 8] and [Figure 7]. The antibacterial activity was ineffective in 25 and 50μL and showed a maximum zone of inhibition of 13 mm against Pseudomonas aeruginosa at 100 µg/mL concentration of the extract.[12]
Antifungal activity by agar well diffusion method
The KE extract was tested for its antifungal activity against three Candida species [Figure 8] and [Table 9]. The antifungal activity showed a maximum zone of inhibition of 15 mm against Candida albicans at 100 µg/mL concentration of the extract.
Our study is the first of its kind that demonstrated the anticandidial activity of the DMSO extracts of KE. Such a property could be related to the presence of enzymes and stable cysteine proteases in the oil. Further, the anticandidial activity of oil suggests that it might be effective against other fungal strains as well.[14]
Wound healing assay
Cell migration assay
Test formulation treatment enhanced the cell migration of 3T3 fibroblasts when tested by scratch wound assay.[15] After 12 h of treatment with 50 μg of test formulation, the migratory nature of the fibroblast could be seen by microscopic examination [Figure 9]. Complete covering of the wound was observed within 24 h of treatment, similar to that of epidermal growth factor (EGF). This response was not recorded either in the cell culture plate maintained as untreated control or placebo treatment [Figure 10]. | Figure 9: Microphotography of cell migration captured at 48 h after incubation using phase-contrast microscope
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Fibroblast proliferation assay
An increased proliferation of fibroblast in response to treatment with test formulation was observed. The rate of proliferation was directly proportional to the concentration. However, concentration above 50 μg/mL did not greatly influence the proliferation rate. A total of 50% increase in the proliferation of fibroblast in response to test formulation at a concentration of 50 μg/mL was demonstrated, whereas in the positive control, EGF showed an 85% of increase in cell proliferation. Cell proliferation was insignificant in untreated and placebo-treated control [Figure 11].
The present study clearly suggests that test formulation enhances the cell proliferation and migration of 3T3 fibroblasts. Cell proliferation and cell migration are two important events necessary for wound healing. We have chosen 3T3 fibroblast cell line, because the fibroblasts play a major role in the wound healing process, besides keratinocytes. Test formulation contains the DMSO extract of KE, which is known to have several medicinal properties including anti-inflammatory activity. KE has a wound healing effect as evidenced by enhanced cell proliferation and migration.
| Discussion | | |
A well-orchestrated integration of the intricate biological and molecular processes of cell migration and proliferation, extracellular matrix deposition, and remodeling is necessary for the best possible healing of a cutaneous lesion. Cellular reactions to cytokines, growth factors, and inflammatory mediators, as well as to mechanical stresses, must be appropriate and accurate. However, chronic wounds, especially those brought on by diabetes, are less likely to heal in an orderly fashion. Such skin wounds do not cure with existing therapy, which mostly employ dressings with moisture control qualities. Failure to heal is caused by a number of pathogenic defects, from disease-specific inherent faults in blood supply, angiogenesis, and matrix turnover to extrinsic variables brought on by infection and ongoing stress. Nevertheless, there is growing reason for optimism for the treatment of diabetic and other chronic wounds despite these difficulties.
A medication must possess both a soul and a body, just like every other living thing, in order to be effective. When metals and plants are mixed, the soul of the plant is transmitted to the metal, making it work and be absorbed by the body. During the burning (heating) process, the plant’s physical structure is lost. While the mineral component serves as a body or vehicle, the medicinal efficacy of the finished medicine is attributed to the plants utilized in the process. After burning, the final product “Kandhaga Ennai” can be used both internally and externally because the leaves of Piper betle and the oil of Brassica nigra serve as a soul for the body (sulfur).
The few analytical parameters of KE reveal the changes in color and pH. It is observed that the oil becomes more acidic after preparation. This formulation also has essential elements that maintain the normalcy of the body counteracting the disease progression, that is, the calcium fastens the wound healing and potassium prevents its undesired activity. The chloride and carbonate ions help in maintaining the cellular integrity by governing the acid–base balance of the cell.
The FTIR results show the observed water O-H stretch, O-H stretch, H-C-H stretch, C=O stretch, N-H stretch, C-C=C symmetric stretch, H-C-H bend, C-O stretch, C -H bend, and C-C stretch, which indicate that the presence of functional groups amide, phenols and alcohols, alkanes, aldehyde, amine, alkenes, alkanes, ester, ether, and alkyne.
The drug is a herbomineral drug with sulfur as the sole mineral ingredient. The remaining ingredients are herbs. The heavy metal analysis infers that all the toxic heavy metals are within range, and the calcium, phosphorous, and sulfur are also within the admissible level. With this, Siddhar’s admirable formulas once again proved that the toxic minerals were converted into almost nontoxic medicines.
The results of the present study indicate that KE has significant antioxidant activities to reduce the harmful effect of radicals particularly caused by oxidative stress. This provides promising guideline regarding the potential uses of this drug in the prevention of age-related disease development but has a positive impact on health and cancer prevention.
Human red blood cell membrane stabilization has been used as a method in estimating the anti-inflammatory property as they are similar to these lysosomal membrane components. The results between diabetic and antidiabetic blood show the differential activity of KE.
Electrostatic contact may easily draw negatively charged bacteria to positively charged mineral surfaces. As a result, minerals included in medications in their oxidized state (metal oxides) may cause damage to bacterial cell walls and enhance permeability to other substances. Overall, it may be hypothesized that a variety of processes work together to prevent microbes from growing and to cause their cells to die. Furthermore, the research medication KE’s antibacterial action may have different pathways depending on the chemistry of the minerals and metals.
The antifungal activity of these plant extracts can be ascribed to the fungicidal action of more than 200 different chemicals, including allicin, allin and volatile sulfur compounds. The use of KE extracts has proven to be more effective than the original standard antifungal drug used.
KE has a definite effect in promoting migration and proliferation of fibroblasts. Moreover, the intake of this oil is an encouraging formulation mode for the delivery of polyherbal actives in the management of superficial wounds and diabetic foot ulcers.
| Conclusion | | |
The study results substantiates the Siddha literature and claim toward the effectiveness of KE for “Ranamegangal” (diabetic wounds) with scientific evidence through anti-inflammatory, antimicrobial, antioxidative, and wound healing potential. The drug standardization provides a quality check for KE, which favors the reproducibility of the product at a larger scale. This study would enable further preclinical animal studies and clinical studies of the drug for healing diabetic ulcers. Thus this drug can be taken to the next level by carrying out studies to know the mechanism of action for the therapeutic effect.
Acknowledgements
Our sincere thanks to the Management, Principal, Vice Principal, and Guide of Velumailu Siddha Medical College and Hospital, Sriperumbudur.
Financial support and sponsorship
This work was financially supported by Central Council for Research in Siddha (CCRS), Ministry of AYUSH, Government of India, New Delhi.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]
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