QUANTIFICATION OF URSOLIC ACID AND MINERALS FROM HEDYOTIS HERBACEA LINN.

QUANTIFICATION OF URSOLIC ACID AND MINERALS FROM HEDYOTIS HERBACEA LINN.

K. Anand Babu ^{* 1}, S. Sivakrishnan ² and S. Jasemine ¹

College of Pharmacy ¹, Kannur Medical College, Anjarakandy - 670612, Kerala, India.

Department of Pharmacy ², Faculty of Engineering and Technology, Annamalai University, Annamalai Nagar, Chidambaram - 608002, Tamil Nadu, India.

ABSTRACT: The whole plant of Hedyotis herbacea Linn. which was claimed to have wound healing activity, was evaluated for phytoconstituent ursolic acid determination. The literature review suggests that ursolic acid was scientifically validated for wound healing activity. So the research work aims to screen the various extract of H. herbacea to determine the ursolic rich fraction. So a high-performance thin-layer chromatographic method was developed for quantitative estimation of ursolic acid in H. herbacea family Rubiaceae. The separation was performed on silica gel 60 F254 HPTLC plates using toluene: ethyl acetate: formic acid (7:3:0.1) as mobile phase for elution of markers from the extract. The determination was carried out by using densitometric absorbance-reflection mode at 516 nm for ursolic acid, and also trace elements such as Magnesium, Calcium, Manganese, Potassium, Iron, and zinc which will favor the wound healing activity, were determined by High-performance liquid chromatography.

Wound healing, Ursolic acid, HPTLC, Trace elements and Vitamins

INTRODUCTION: Wound healing is an intricate process in which the tissue repairs by itself after an injury ¹. It is a process that involves the activation of intercellular pathways, coordination of tissue integrity, and homeostasis. The wound is actually a “disruption of normal anatomic structure and function”, according to the wound healing society ². Wound healing is a natural biological process that is a combination of a series of independent processes where dermal cells and epidermal cells, ECM, plasma-derived proteins, growth factors, and cytokines all act together to initiate wound healing ^{3, 4, 5}. Depending upon the nature and depth of the injury, the wound healing can be categorized ⁶.

Wound healing is categorized into four phases such as the hemostasis phase, the inflammatory phase, the proliferation phase, and the remodeling phase. For successful healing of a wound, all these four phases must take place in the correct sequence and time frame. Many factors can obstruct one or more phases of this process, thus causing improper or impaired wound healing. It was reported that Non-healing wounds affect about 3 to 6 million people in the United States, with persons 65 years and older with 85% of these events. Non-healing wounds will result in enormous health care expenditures, with the total cost estimated at more than $3 billion per year ^{7, 8}.

The factors that influence the repair of wounds can be categorized into local and systemic factors. Local factors such as oxygenation, infection, foreign body, venous insufficiency. Systemic factors include age and gender, sex hormones, stress, ischemia, diseases: Diabetes, Keloids, Fibrosis, hereditary healing disorders, jaundice, uremia, Obesity, Alcoholism and smoking, cancer, and Nutrition. Medications like chemotherapeutics, NSAIDs, and glucocorticoid steroids can also retard wound healing ⁹.

For more than 100 years, nutrition has been observed as a very crucial factor that affects wound healing. Energy, carbohydrate, protein, fat, vitamin, and mineral metabolism can affect the healing process ¹⁰. For optimal repair, several micro-nutrients are also essential. Sequential events in wound repair require a conducive environment within the wound bed and a balanced pool of metal ions, of which at least calcium, zinc, magnesium, copper, manganese, iron, sodium, and potassium are important ¹¹. It was suggested that iron indirectly stimulates HDF (Human Dermal Fibro-blasts) ECM (Extracellular Matrix) deposition via modulation of macrophage behavior, with potential therapeutic application in pathologic healing ^{12, 13}.

An analysis of wound exudates, it showed that elements such as magnesium promote tissue adhesion, migration of macrophages, keratinocytes, fibroblasts, and production of type I collagen, all of which contribute to the wound healing process ^{14, 15, 16}. Regulation of ion channels by KCl causes upregulation of TSP1 (Thrombospondin 1) protein, which is involved in angiogenesis and wound healing ¹⁷. KCl-treated cells exhibited induction of TSP1 levels, which would lead to anti-angiogenic effects. It has been reported that clinical studies on the treatment of breast fissures with manganese salts with an improvement of wound healing ^18, which might be due to the induction of pro-MMP-9 (Matrix metalloproteinase), stimulating keratinocyte migration via αvβ6. Expression of the MT gene is upregulated in the skin following topical application of zinc and in wound margins, particularly in regions of high mitotic activity. This induction of MT in the wound margin may reflect its role in promoting cell proliferation and reepithelialization ¹⁹.

Study ²⁰ revealed that calcium alginate acted as a promoter of diabetic wound healing via increasing the contraction of wounds, attenuating inflammatory reaction by increasing the levels of type I collagen and hydroxyproline, leading to a significant increase in the wound tensile strength. The accelerating effect of calcium alginate was due to the stimulation of collagen I expression and elevation of collagen I to III ratios to a large extent.

Herbal medicines are widely accepted because of their various advantages like lesser side effects, safety, and efficacy which ultimately increases the compatibility with humans ²¹. Oldenlandia herbacea Roxb. (H. herbacea) (family- Rubiaceae) is an erect, glabrous annual shrub found in temperate and tropical regions of Africa and Asia. Its extract or decoction has been reported to be useful in the treatment of malaria. The decoction of the herb has been used for bathing rheumatic patients, and the powdered herb administered with honey for rheumatic fever and swellings. The herb is boiled in oil, and the oil is used for elephantiasis and pains in the body. The leaves have been employed as an expectorant in Asthma ²².

Ethnomedically, this plant is recognized as one of the ingredients in Jaundice treatment. A paste of leaves is considered as emollient and applied to abscesses and wound ^23-24. The plant is also reported to use in deafness.

FIG. 1: HEDYOTIS HERBACEA

This research work attempts to estimate the ursolic acid in various extracts of H. herbacea by HPTLC, which was proved to have a wound healing activity ^25, and also estimate the minerals and vitamins which favors the wound healing activity. The electronic image of chromatographic fingerprint and densitogram for detecting the presence of marker compound in the sample can be provided by HPTLC ²⁶.

H. herbacea contains an interesting triterpenoid of nature, ursolic, acid as it possesses many beneficial effects such as anti-inflammatory, hepatoprotective, antibacterial, antiulcer, etc. It provoked us to identify and determine the presence of ursolic acid in the leaves also. The ursolic acid is a triterpenoid that is derived from the subgroup named ursane ²⁷.

Moreover, trace elements like Zinc (Zn), Magnesium (Mg), Manganese (Mn), Iron (Fe), Calcium (Ca) and Potassium (K) supports wound healing property as essential trace mineral are required for cellular growth and replication. A survey of available literature showed that there was no report available on the trace element content of the leaves. So it decided to estimate the trace element content of the leaves.

MATERIALS AND METHODS:

Chemicals: Petroleum ether, Chloroform, Ethyl acetate, and Ethanol were purchased from Merck (India). Standard of ursolic acid (99% purity) were purchased from Sigma (New Delhi, India).

Plant Material:  Fresh leaves of the plant H. herbacea were collected from the Tirunelveli district, Tamil Nadu, India. The specimen was authenticated by V. Chelladurai, Research officer-Botany (Scientist-C), Centre Council for Research in Ayurveda & Siddha, Government of India. A voucher specimen no is (CS-BOT-HH01).

Preparation of Leaves Extract: Leaves were dried in shade and ground to obtain a fine powder. The crude powder was subjected to extract with various solvent such as Petroleum ether, chloroform, ethyl acetate, and ethanol. The crude powder was kept in contact with respective solvents for 7 days at room temperature with occasional shaking by the maceration technique. Then the extract was filtered, and the filtrate was concentrated at reduced pressure to obtain the residue. Then the residue of H. herbacea was subjected to HPTLC estimation.

Development of HPTLC Fingerprint:

Instrument: Linomat 5 applicator (CAMAG, Switzerland), twin trough chamber (20 × 10 cm; CAMAG, Switzerland), TLC scanner IV (CAMAG, Switzerland), winCATS version 1.4.6 software (CAMAG, Switzerland), microsyringe (Linomat syringe 659.0014, Hamilton-Bonaduz Schweiz, CAMAG, Switzerland), UV chamber (CAMAG, Switzerland), and precoated silica gel 60 F254 aluminium plates (20 × 10 cm, 100 μm thickness; Merck, Darmstadt, Germany) were used in the study.

Sample: The dried extracts of petroleum ether, Chloroform, Ethyl acetate, and Ethanol were separately dissolved in respective solvents and made up to 10mg/ml concentration.

Stationary Phase: The HPTLC plates Silica gel 254, thin-layer chromatography (20 cm × 10 cm).

Mobile Phase: The standard ursolic acid stock solution was prepared in the mobile phase toluene: ethyl acetate: formic acid (7:3:0.1) in the concentration of 100mcg/ml.

Detection Wavelength: Different amounts of (200, 400, 600, 800, and 1000ng) ursolic acid and various residues of extracts (200ng) were loaded onto a TLC plate and scanned at 516nm to get the calibration curve in the ascending mode.

RESULTS AND DISCUSSION: Various proportions of toluene and ethyl acetate were tried as the mobile phase on silica gel HPTLC plates, and a ratio of (7:3:0.1) provides a good elution. The calibration curve of ursolic acid was prepared by plotting the concentration of ursolic acid versus the area of the peak was depicted in Fig. 5.

Petroleum ether, chloroform, ethyl acetate, and ethanolic extract of H. herbacea were analyzed by the optimized chromatographic condition of HPTLC analysis which showed 4mg/g of ursolic acid in chloroform extract as mentioned in Fig. 2 and 4, Table 1 and other extracts doesn't show the presence of it. The screened chloroform extract was subjected to trace element, and vitamin content analysis by HPLC as listed in Tables 2 and 3.

FIG. 2: QUANTITATIVE ESTIMATION OF URSOLIC ACID AT 516 nm (AT WHITE LIGHT)

TABLE 1: ESTIMATION OF URSOLIC ACID IN H. HERBACEA

TABLE 2: LIST OF MINERALS PRESENT IN H. HERBACEA

TABLE 3: LIST OF VITAMINS PRESENT IN H. HERBACEA

FIG. 5: STANDARD CALIBRATION FOR URSOLIC ACID IN ALL TRACK (CONCENTRATION VS. AREA)

FIG. 6: HPTLC ESTIMATION OF URSOLIC ACID (3D DISPLAY)

CONCLUSION: The proposed HPTLC method was used for the quantitative estimation of ursolic acid. Ursolic acid is one of the factors that supports wound healing, and also the nutritional factor which promotes the wound healing activity was also estimated by HPLC. The HPTLC estimation of ursolic acid confirms its presence in chloroform extract of H. herbacea , which is having a significant effect on wound healing. The extract also indicated the presence of various minerals such as zinc, calcium, magnesium, manganese, potassium and iron at different concentrations which thereby provides an added advantage along with ursolic acid in wound healing activity, since all the minerals are reported for their significance in wound healing activity. Hence based on this report, chloroform extract of H. herbacea will be utilized to evaluate the in-vitro and in-vivo wound healing activity for further confirmation of ursolic acid-enriched extract to prove its efficiency in wound healing.

ACKNOWLEDGEMENT: Nil

CONFLICTS OF INTEREST: There is no conflict of interest.

REFERENCES:

1. Nguyen DT, Orgill DP and Murphy GT: The Pathophysiologic Basis for Wound Healing and Cutaneous Regeneration. Biomaterials for Treating Skin Loss. Berlin, Germany, Elsevier; 2009: 25‑
2. Anuroop UP, Nair SS and Chacko AJ: Development and evaluation of medicated biodegradable film for wounds and burns. Int J Pharm Sci & Res 2019; 10(12): 5664-72.
3. Metcalfe AD and Ferguson MW: Skin stem and progenitor cells: Using regeneration as a tissue‑engineering strategy. Cell Mol Life Sci 2008; 65: 24‑
4. Trent JT and Kirsner RS: Wounds and malignancy. Adv Skin Wound Care 2003; 16: 31‑
5. Troxler M, Vowden K and Vowden P: Integrating adjunctive therapy into practice: the importance of recognising ‘Hard‑To‑Heal’ wounds. World Wide Wounds
6. Zhong SP, Zhang YZ and Lim CT: Tissue scaffolds for skin wound healing and dermal reconstruction. Wiley Interdisc Rev Nanomed Nanobiotechnol 2010; 2: 510‑
7. Mathieu D, Linke JC and Wattel F: Non-healing wounds. In: Handbook on hyperbaric medicine, Springer, 2006: 401-27.
8. Menke NB, Ward KR, Witten TM, Bonchev DG and Diegelmann RF: Impaired wound healing. Clin Dermatol 2007; 25: 19-25.
9. Kumar N, Singh A, Sharma DK and Kishore K: Wound healing activity of methanolic extract of Arnebia benthamii. Int J Pharm Sci & Res 2020; 11(1): 328-33.
10. Arnold M and Barbul A: Nutrition and wound healing. Plast Reconstr Surg 2007; 117(7 Suppl): 42S-58S.
11. Lansdown ABG, Sampson B and Rowe A: Sequential changes in trace metal, metallothionein and calmodulin concentrations in healing skin wounds. J Anat (London) 1999; 195: 375-86.
12. Ploeger DT, Hosper NA, Schipper M, Koerts JA, de Rond S and Bank RA: Cell plasticity in wound healing: paracrine factors of M1/M2 polarized macrophages influence the phenotypical state of dermal fibroblasts. Cell Commun Signal 2013; 11: e29
13. Jetten N, Roumans N, Gijbels MJ, Romano A, Post MJ, de Winther MP, van der Hulst RR and Xanthoulea S: Wound administration of M2-polarized macrophages does not improve murine cutaneous healing responses. PLoS One 2014; 9: e102994
14. Wayne K: Physiology and healing dynamics of chronic cutaneus wounds. Am J Surg 1998; 176: 26-38.
15. Jensen L: Collagen, scaffold for repair or execution. Cardiovascular Res 1997; 33: 535-53.
16. Witt M and Thornton F: Enhancement of fibroblast collagen synthesis by nitric oxide. NO 2007; 4: 572-82.
17. Shim JH, Lim JW, Kim BK, Soo Jin Park SJ, Kim SW and Choi TH: KCl mediates K⁺ channel-activated mitogen- activated protein kinases signaling in wound healing. Arch Plast Surg 2015; 42: 11-19.
18. Barrat J, Crepin G, Darbois Y and Sirot S: Copper and manganese gluconate in the treatment of breast fissures during breast feeding. Rev Fr Gyn Obstet 1992; 87: 49-51.
19. Molnar J: Nutrition and Wound healing. CRC press Taylor and francis group 200.
20. Wang T, Gu Q, Zhao J, Mei J, Shao M, Pan Y, Zhang J, Wu H, Zhang Z and Liu F: Calcium alginate enhances wound healing by up-regulating the ratio of collagen types I/III in diabetic rats. IJCEP 2015; 8(6): 6636-45.
21. Koparde AA, Doijad RC, Patil A and Magdum CS: Isolation and quantification of gallic acid from Eulophia ochreata by HPTLC. IJPSR 2020; 11(8): 3859-66.
22. Kirtikar KR and Basu BD: Indian Medicinal Plants. Bishen Sing Mahendra Pal Sing, Dehradun, India 1987; 2: 1264-65.
23. Pullaiah T: Encyclopedia of world medicinal plants 1: 1064.
24. Indian Medicinal Plants: A Compendium of 500 Species 1994; 3: 124-126.
25. Pravez M and Patel AK: Wound healing activity of ursolic acid stearoyl glucoside (UASG) isolated from Lanata camara IJPSR 2014; 5(10): 4439-44.
26. Bhagure LB and Une HD: Identification and quantification of oleanolic acid from Cyclea peltata by high performance thin layer chromatography (HPTLC) method. Int J Pharm Sci & Res 2019; 10(11): 4899-06.
27. Sanaye MM and Zehra S: Development and validation of HPTLC method for identification and quantification of ursolic acid in the leaves of Alstonia scholaris. Int J Pharm Sci & Res 2020; 11(11): 5540-46.
    

    ---

    How to cite this article:

    Babu KA, Sivakrishnan S and Jasemine S: Quantification of ursolic acid and minerals from Hedyotis herbacea Linn. Int J Pharm Sci & Res 2021; 12(7): 3839-43. doi: 10.13040/IJPSR.0975-8232.12(7).3839-43.

    ---

    All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---