In vitro – In vivo Cellular Reprogramming and Antioxidant potential of Herbal Drug: Fumaria officinalis

Ruchi Khare*, Neeraj Upmanyu

School of Pharmacy and Research, People's University, Bhopal (M.P.) India 462037

*Corresponding Author E-mail: ruchiapharma@gmail.com

ABSTRACT:

Context: The medicinal plants have numerous pharmacological properties and having lesser side effects. Today there is increasing trend of medicinal plants as anti-aging and anti-wrinkle agents. Objective: The aim of this study is to evaluate antioxidant, anti-aging and anti-wrinkle potential of Fumaria officinalis. Materials and methods: Fumaria officinalis, fumitory (Papaveraceae) is native to Europe and Asia, now grows wild in parts of North America. Powdered crude drug 100 g were successively extracted in a soxhlet apparatus with petroleum ether (60-80ºC), chloroform and methanol. After successive solvents extraction methanolic extract was used for testing of antioxidant potential using DPPH assay. Further, anti-aging potential of extract was investigated by inhibitory effect of various enzymatic estimations i.e. Col-I, Ela-I and Hya-I inhibitory assays on early aging human skin fibroblasts. Anti-wrinkle potential of plant Fumaria officinalis was done by using UV light induced photo aging model. Results: Phytochemical analysis showed the presence of glycosides, alkaloids flavonoids, and triterpenoids, saponins and Phenolic compounds in high level. Extract showed inhibitory concentration (IC₅₀:35.5) and ascorbic acid the standard antioxidant showed inhibitory concentration (IC₅₀:20.10). In enzymatic estimations assay, the Col-I, Ela-I and Hya-I of extract were assessed showing inhibitory concentration as Col-I (IC₅₀:41.25), Ela-I (IC₅₀:45.31) and Hya-I (IC₅₀:30.55) respectively. Thus, MeOH extract of Fumaria officinalis able to inhibit 50% of the activity of aging related enzymes Col-I, Ela-I and Hya-I. The wrinkle score of negative control i.e. UV treated group was 2.83±0.408 and MeOH extract of Fumaria officinalis treated group is 2.33±0.516. Conclusion: This study concluded that MeOH extract of Fumaria officinalis has confirmed the high antioxidant potential and In vitro and In vivo inhibitory potential of antiaging enzymes assessed, thus they could be used for further development of cosmetic products and nutraceuticals.

KEYWORDS: Fumaria officinalis, peet papra, photo aging, wrinkle score, nutraceuticals.

1. INTRODUCTION:

Medicinal plants are gift of God, to cure infinite number of diseases in human beings and other living organism. These Plant materials have been extensively used in the indigenous system of medicine which is mention in the Ayurveda and other Indian literature.

They have been the major source of drugs in all system of medicine and other ancient systems in the world. Such plants kingdom harbors an in exhaustible source of active ingredients invaluable in the management of many intractable diseases.

Skin aging is the natural process due to photo aging by environmental factors such as chronic UV radiation. The repetitive exposure to UV radiation cause accelerated physical changes in the skin and connective tissue through the formation of lipid peroxides, the cell contents and Reactive Oxygen Species (ROS)^[1]. It leads to loss of skin elasticity implicated in formation of wrinkle, uneven pigmentation, brown spots, laxity and leathery appearance, solar elastosis, actinic purpura, precancerous lesions, skin cancer, and melanoma^[2-4]. During aging process, collagen, elastin, and hyaluronic acid decrease, that causes loss of strength and flexibility in the skin, resulting in visible wrinkles. It is also related to increasing enzymes activity including collagenase, elastase and hyaluronidase. Collagenase is known as an enzyme that plays role in the degradation of collagen. Collagen is the main component with percentage of 70-80 % of the total skin weight, the increasing degradation of collagen is significant in the photo aging process^[5,6]. Hyaluronan or hyaluronic acid is one of important components of the tissue matrix substance and has a role in the development, growth, and repair of damaged tissue^[6]. Meanwhile, elastin play a role in the maintenance of skin elasticity, but elastase can degrade it^[7]. Degradation of the Extracellular Matrix (ECM) has been directly linked to skin aging and is correlated with an increase in activity of certain enzymes involved in skin aging^[8,9]. Inhibition of these enzymes is crucial in anti aging prevention^[10]. It has been reported that skin aging occurs in the presence of cumulative endogenous damage due to Reactive Oxygen Species (ROS)^[11].

The Siddha and Ayurveda systems of treatment are being increasingly recognized as an alternate approach for the antiaging & antiwrinkle treatment. Traditional herbs provide interesting and largely unexplored sources for the development of potential new cosmetic and pharmaceutical products. In this regard, one such plant is Fumaria officinalis. Fumitory has been highly valued since at least Roman times for its tonic and blood cleansing effect upon the body. No scientific report is available to date to validate the antiaging and antiwrinkle property of Fumaria officinalis.

Extracts of Fumaria officinalis (Fumariaceae) have been traditionally used for treatment of some skin diseases (rashes or conjunctivitis), rheumatism, stomach ache, abdominal cramps, fever, diarrhea, syphilis and leprosy ^[12]. Phytochemical effects of Fumaria officinalis related to several alkaloids (adlumidiceine, copticine, fumariline, perfumine, protopine, fumaranine, fumaritine, paprafumicin and paprarine)^[13], flavonoids (isovitexin, rutin, isoquercitrin and quercitrin)^[14]. Fumaria officinalis possess antihelmintic, antipyretic and hypoglycemic properties^[15-17] reported in earlier studies.

2. MATERIAL AND METHODS:

2.1. Preparation of plant material:

The leaves of Fumaria officinalis plant collected from the nearby area of Bhopal, Madhya Pradesh, India. The plant was identified and authenticated by Dr. Zia Ul Hasan, Professor& Head-Department of Botany, Safia college of Science, Bhopal. A voucher specimen number 125/Bot/Saf/17 was kept in Department of Botany, Safia college of Science, Bhopal for future reference.

2.2 Extraction of plant material:

Plant was shade dried and ground (1mm) using a blender. Powdered crude drug 100g was successively extracted in a soxhlet apparatus with petroleum ether (60-80ºC), chloroform and methanol. All the extracts were concentrated by distillation of the solvent and evaporating them under vacuum at 40°C and stored at 4°C until use. The extract obtained with each solvent was weighed to a constant weight and percentage w/w basis was calculated as percentage yield. The extract yield was determined gravimetrically (Table 1).

Weight of extract (g)

% Yield= ––––––––––––––––––––––––––––––

Weight of dry powder (g) ×100

Table 1: Percentage yield

Scientific Name	Plant Part	Percentage yield
Fumaria officinalis	Leaves	3.88

2.3 Chemicals:

All the chemicals used were of analytical grade and were obtained from Merck, sigma and S.D. Fine Chemicals.

2.4 Qualitative Phytochemical Screening:

Detailed qualitative phytochemical analysis was performed to identify presence or absence of different Phytoconstituents as phenols, steroid/ triterpenoids, saponins, tannins, glycosides, flavonoids, and alkaloids^[18,19].

2.5 Selection of animals:

The study was carried out after obtaining the Institutional Animal Ethics Committee approval number PBRI/IAEC/PN-17037a of CPCSEA from Pinnacle Biomedical Research Institute. Swiss albino mice were used in this experiment. Animals were housed in poly acrylic cages maintained under standard conditions of 18°C±2°C and 12 h light/ dark cycle. Animals had free access to standard pellet diet and water, ad libitum.

2.6 Antioxidant activity:

2.6.1. DPPH (2, 2-Diphenyl-1-picrylhydrazil) radical scavenging assay:

DPPH is a stable organic radical which has the capacity to scavenge biological reagents. Its solution is deep purple in color with an absorption peak at 517 nm, which disappears with the presence of the radical scavenger in the reactive system, when odd electrons of nitrogen in DPPH molecule are paired. The reactive rate and the ability of the radical scavenger depend on the rate and the peak value of disappearance of the DPPH^[20,21]. 2ml of DPPH radical solution (75μM) was prepared and 2ml solutions of plant extract of various concentrations (20 μg/ml - 100μg/ml) were added and ascorbic acid was used as standard. The reaction mixtures were shaken thoroughly and kept at dark for 30 min. Control solution was prepared by adding 2ml of methanol with 2ml of DPPH solution. The absorbance of all the reaction mixtures and control solution was measured at 517nm. The % Inhibition was calculated using following formula:

AC 517 nm- AS 517 nm

% Inhibition = –––––––––––––––––––––– x 100

AC 517 nm

Where, AC is absorbance of Control and AS is the absorbance of Sample. The inhibition concentration (IC-50) value was determined from extrapolating the graph of % Inhibition versus the concentration of extract (using linear regression analysis), which is defined as the amount of antioxidant necessary to reduce the initial radical concentration by 50%. Lower the IC-50 value higher the antioxidant effects.

2.7 In-vitro Enzymatic Estimation:

2.7.1. Hyaluronidase Inhibitory Activity Assay:

A mix of 25μL of various level of samples (20-100μg/ mL) of plant extracts were prepared and 3μL hyaluronidase from bovine serum albumin[Sigma A4503] was pre-incubated for 10 min at 37°C and then added 12μL phosphate buffer (300mM, pH 5.35) for 10 min at 37°C. Afterward, 10μL hyaluronic acid substrate [Sigma H5388, USA] was added and incubated for 45 min at 37°C. Decomposition reaction of hyaluronic acid was stopped by adding 100μL acidic albumin acids. Mixed solution incubated at room temperature for 10 min, and then absorbance was measured at 600 nm wavelengths^[22].

(1- Absorbance _Control) X 100

% Inhibition = –––––––––––––––––––––––––––

Absorbance _sample

2.7.2. Collagenase Inhibitory Activity Assay:

Collagenase is an enzyme in the matrix metalloproteinase family. Collagen is an abundant structural protein present in the connective tissue of animals.

50mM Tricine buffer (400 mM NaCl and 10mM CaCl2, pH 7.5). Collagenase from Clostridium histolyticum was dissolved in the buffer for use at an initial concentration of 0.8units/ml. The synthetic substrate, FALGPA, was dissolved in the Tricine buffer to 2mM. Sample extracts were incubated with the enzyme in the buffer for 15 min before adding substrate to start reaction. The final reaction mixture (75μl total volume) contained 25μl of 50mM Tricine buffer, 25μl of test extract (20–100 μg/ml), and 25μl of 0.1 units of enzyme Collagenase. Controls performed with 50mM Tricine buffer as test extracts were dissolved in Tricine buffer (50 mM), while rutin was used as a positive control. After adding 50μl of 2mM FALGPA substrate, collagenase activity was measured immediately at 340 nm^[23].

(1- Absorbance _Control) X 100

% Inhibition = –––––––––––––––––––––––

Absorbance _sample

2.7.3. Elastase Inhibitory Activity Assay:

This assay was performed in 0.2mM Tris-HCL buffer (pH 8.0). Porcine pancreatic elastase was dissolved to make a 1 mg/ml stock solution in 0.2mM Tris-HCL buffer. The substrate N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA) was dissolved in buffer at 0.8mM. The test extracts (20-100mg/ml) were incubated with the enzyme for 20 min before adding substrate to begin the reaction. The final reaction mixture (Total 250μl) contained 50μl plant extract, 160μl buffer, 20μl enzymes, and 20μl substrate. Rutin was used as a positive control. Negative controls were performed using Tris-HCL buffer. Absorbance was measured immediately at 410 nm and then continuously for 20 min using a 96 well micro plate reader^[24].

(1- Absorbance _Control) X 100

% Inhibition = –––––––––––––––––––––––––

Absorbance _sample

2.8. Skin irritation study:

Female Swiss albino mice, weighing 15–25g, were used. 24h before the test (dose application), the hair on demarcated areas of approximately 4 cm² on the dorsal surface of each mouse was removed using a Rose Anne French hair- removing cream. The mice were observed for 48 h and those showing any abnormal hair growth or any reaction to the cream were excluded. Hair removing cream was preferred to shaving blade in order to minimize free radical production due to trauma from the blade.

The final cream formulation was evenly applied to 1 cm2 area of each rabbit. Skin reaction at the site of application was subjectively assessed and scored once daily at zero day, 24 hrs, 48 hrs and 72 hrs (post test observation period) accordingly.

2.9. In- vivo Assay:

UV light exposure conditions and development of photo-ageing:^{[25, 26]}

The mice were divided into 3 groups of 18 animals each. Group I served as control. Group II animals received 5 min UV exposure twice a day and served as irradiated control. The test groups III received both UV radiations and Fumaria officinalis extract topically, respectively. The treatment was given 4 h prior to UV exposure as per the protocol. All the animals were kept inside a solar simulator (designed in the laboratory and fitted with UV lamp) at a distance of 40 cm from the UV light source (Ultra Vitalux 300 W Waton® bulb, Germany). The bulb gave the full spectrum of UV radiation, i.e., 260 - 400 nm, simulating the full solar spectrum. UV exposure was controlled by time of exposure. Exposures were given twice daily for 5 min. For the mice receiving topical formulation treatment, the dorsal skin was treated with formulation 4 h prior to each UV radiation exposure. The test sample was delivered as topically at a dose of 100 μl/cm2 area of the skin with micropipette. The animals were treated for 30 days following which the skin was excised and used for biochemical estimation and histological study. Skin Slides of mice were obtained after termination of experiment and fixed in 4% paraformaldehyde for 24 h. Then skin specimens were embedded in paraffin and sectioned. Hematoxylin and eosin (H&E) staining was used to observe epidermal thickness. H&E staining was successively conducted by deparaffination, hydration, hematoxylin staining, eosin staining, and dehydration. Later, observed by microscopy. The wrinkle score was observed at the termination of the study^[24].

3. RESULTS AND DISCUSSION:

3.1 Phytochemical Screening of Fumaria officinalis

Phytochemical screening of the plant showed the presence of carbohydrates, proteins, glycosides, phenols, flavonoids, terpenoids, saponins, and alkaloids. The result of Fumaria officinalis phytochemical screening can be seen in Table 2.

Table 2: The result of qualitative phytochemical screening of Fumaria officinalis

Phytochemical content	*Fumaria officinalis*
Glycosides	+
Triterpenoids	+
Flavonoids	+
Phenols	+
Saponins	-
Alkaloids	+
Tannins	+
Steroids	+

3.2 DPPH (2, 2-Diphenyl-1-picrylhydrazil) radical scavenging assay

The in-vitro antioxidant and enzymatic activities of the Fumaria officinalis was studied. Free radicals can damage the skin by altering the lipid contents in cellular membranes and by affecting the passage of nutrients and other active components in cell structure. The major protein content of healthy functional collagen and elastin fibers also get altered thus resulting in formation of wrinkles, sagging and loss of skin tone. The enzymes produced by collagen are attacked by free radicals. Reactive oxygen species play a complex role in inflammatory cascade^[27]. UV irradiation generates reactive oxygen species that can induce expression of the Matrix Metalloproteinase (MMP) family, which degrades collagen and other Extracellular Matrix (ECM) proteins that consist of the dermal connective tissue^[28].

Table 3: IC₅₀ value DPPH scavenging activity of Ascorbic acid and Fumaria officinalis

Concentration	Control	% Inhibition
Concentration	Control	Ascorbic acid	*Fumaria officinalis*
20 µg/ml	0.632	54.97	45.89
40 µg/ml	0.632	61.09	52.22
60 µg/ml	0.632	67.76	54.91
80 µg/ml	0.632	73.66	60.28
100 µg/ml	0.632	78.58	63.13
IC₅₀		20.10	35.33

Graph 1: Percentage Inhibition of Ascorbic acid and Fumaria officinalis by DPPH Assay

The IC₅₀value of ascorbic acid for DPPH is 20.10 and extract of Fumaria officinalis showed the higher IC₅₀35.33 (Table 3). Graph 1 shows the percent inhibition values for DPPH scavenging activity assay. Thus, the extract of Fumaria officinalis exhibited antioxidant activity when compared to standard antioxidant ascorbic acid. Lower the IC₅₀ stronger the antioxidant activity means activity of plant is less but close to the standard ascorbic acid.

3.3 Hyaluronidase Inhibitory Activity Assay:

The polysaccharide hyaluronan (hyaluronic acid, HA) was isolated from bovine vitreous humor by Meyer and Palmer, 1934. Hyaluronidase assay based on precipitation of HA with cetylpyridinium chloride, which is used for high throughput screening for hyaluronidase inhibitors. This method can be used to evaluate anti aging activity of various herbal formulations^[29].

Table 4: Hyaluronidase inhibitory activity of Rutin and Fumaria officinalis

Concentration	Control	% Inhibition
Concentration	Control	Rutin	*Fumaria officinalis*
20 µg/ml	0.976	51.53	47.54
40 µg/ml	0.976	57.17	53.18
60 µg/ml	0.976	60.86	58.91
80 µg/ml	0.976	70.38	64.65
100 µg/ml	0.976	78.17	75.82
IC₅₀		19.06	30.55

Graph 2: Percentage Inhibition of Rutin and Fumaria officinalis by Hyaluronidase assay

Based on table 4 extract of Fumaria officinalis showed the IC₅₀30.55 which is almost near about the value observed in standard drug rutin with IC₅₀19.06. The anti-hyaluronidase activity of Fumaria officinalis has not been reported to date.

Graph 2 showed Percentage Inhibition of Rutin and Fumaria officinalis i.e. 78.17 and 30.55 respectively at highest concentration (100µg/ml).

3.4 Elastase Inhibitory Activity Assay:

Elastin is a protein found in connective tissue which is responsible for the elasticity of the skin and lungs. This protein is catalysed by the enzyme elastase. Degradation of elastin by intracellular elastase increases with age and/or repeated UV-radiation, leading to skin aging ^{[30 and 31]}.

Graph 3: Percentage Inhibition of Rutin and Fumaria officinalis by Anti- elastase assay

The elastase inhibitory activity of Fumaria officinalis extract and Rutin were measured and showed in Graph 3. Elastase inhibitory activity of Fumaria officinalis extract and Rutin showed the highest inhibition percentage at the highest concentration (68.36 and 62.204 respectively). However, Rutin showed the highest activity in elastase inhibition with IC₅₀ value 25.11μg/mL. Fumaria officinalis have IC₅₀ value 45.31 (table 5). The results showed that Fumaria officinalis extract posses low elastase inhibition compared to Rutin.

Table 5: Elastase inhibitory activity of Rutin and Fumaria officinalis

Concentration	Control	% Inhibition
Concentration	Control	Rutin	*Fumaria* officinalis
20 µg/ml	0.844	47.512	40.88
40 µg/ml	0.844	53.555	49.88
60 µg/ml	0.844	58.057	53.44
80 µg/ml	0.844	60.308	62.44
100 µg/ml	0.844	62.204	68.36
IC₅₀		25.11	45.31

3.5 Collagenase Inhibitory Activity Assay:

Collagen, the major component of the skin, is degraded by the enzyme collagenase. Inhibition of collagenase activity delays the process of forming pre-collagen fibers and subsequently the wrinkling process ^[32].

Table 6: Collagenase inhibitory activity of Rutin and Fumaria officinalis

Concentration	Control	% Inhibition
Concentration	Control	Rutin	*Fumaria officinalis*
20 µg/ml	0.912	52.741	46.60
40 µg/ml	0.912	56.359	49.34
60 µg/ml	0.912	61.513	53.84
80 µg/ml	0.912	68.969	57.68
100 µg/ml	0.912	71.710	62.17
IC₅₀		11.54	41.25

Graph 4: Percentage Inhibition of Rutin and Fumaria officinalis by Anti-collegenase assay

The Fumaria officinalis extract possess Collagenase inhibitory activity. Collagenase activity of Fumaria officinalis extract at 100 micro/ml is 62.17 and Rutin is 71.71. However IC₅₀ value of Fumaria officinalis extract was 41.45 and Rutin was 11.54.

3.6 Inhibition of UV-induced wrinkle formation by methanolic extract of Fumaria officinalis

The number of wrinkles was measured in the vehicle, UV irradiated and treated mice to determine whether treatment with methanolic extract inhibit wrinkle formation induced by UV radiation or not. After UV radiation, deeper and wider wrinkles were formed and number of wrinkles was significantly (P<0.001) higher in the vehicle-treated group than in the no-irradiation group. In particular, the largest decrease was detected in the treated group of methanolic extract of Fumaria officinalis. Therefore, the topical application of methanolic extract of Fumaria officinalis can effectively inhibit wrinkle formation on the dorsal skin of animal.

Table 7: Estimation of Wrinkle Score

Group	Treatment	Wrinkle score
1	Control	0±0
2	UV treated	2.83±0.408
3	UV + Methanolic extract of Fumaria officinalis	2.33±0.516

Group 1: Normal: control with pellet diet only; Group 2: UV irradiated + standard diet; Group 3: UV irradiated + standard diet + methanolic extract of Fumaria officinalis. Values are means ±SDs (n = 6) ##P < 0.001 normal control versus irradiated control

a) Control group

b) Standard (Rutin 1%)

c) UV Irradiated group

d) UV irradiated + Methanolic extract of Fumaria officinalis

Figure: 1 Photomicrography of skin

3.7 Histopathological study:

The changes in histological structures of mouse skins were observed in the normal, UV irradiated and extract treatment group. UV treated group showed thicker epidermis and dermis layer as compared to normal control group. Adipose tissues were also found in higher level in subcutaneous region as compare Fumaria offinalis extract treated group. From these results it is found that the topical application of methanolic extract of Fumarai officinalis can induce a decrease in the thickness of epidermis and dermis, and the number of adipose tissues in skin.

4. CONCLUSION:

This is the novel study in which herbal extract of Fumaria officinalis was used to investigate the anti-elastase, anticollagenase and anti-hyaluronidase activity. The free radical scavenging activity and enzyme inhibitory activity of the plant extract suggests that they can help restore skin elasticity and thereby slow the wrinkling process and will be subjected to further testing and isolation of the active compounds. UV radiations damage the skin tone by damaging macromolecules such as proteins and lipids in skin. Commercially available chemical based sunscreens prepared by using titanium di-oxide, zinc oxide, di -oxybenzone etc. thus may cause dermatitis and skin irritation by reacting with others molecules. Therefore, it is the need of today’s life style to prepare such herbal preparations inclusive of polyphenols, flavonoids and antioxidants for better tolerability and greater efficacy.

5. LIST OF ABBREVIATIONS:

UV- ultraviolet radiation

ECM -extracellular matrix

SC- stratum corneum

MeOH- methanolic

DPPH-2, 2-Diphenyl-1-picrylhydrazil

AC- absorbance of Control

AS - absorbance of Sample

Col-I- Collagenase enzyme

Ela-I-Elastase enzyme

Hla-I- Hyaluronidase enzyme

FALGPA- (N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala)

SANA - N-Succinyl-Ala-Ala-Ala-p-nitro anilide

GAGs- Glycos amino Glycan

MMP - Matrix Metalloproteinase

FO- Fumaria officinalis

HA - hyaluronic acid

IC- inhibition concentration

H&E- Hematoxylin and eosin

CPCSEA- Committee for the Purpose of Control and Supervision of Experiments on Animals

6. ACKNOWLEDGEMENT:

I whole heartedly thanks to Dr. Neeraj Upmanyu, Mentor, Dr. Megha Jha, and Mr. Anurag Shrivastava who contributed to my work.

7. CONFLICT OF INTEREST:

We declare that we have no conflict of interest.

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Received on 20.05.2019 Modified on 15.06.2019

Research J. Pharm. and Tech. 2019; 12(11):5517-5523.

DOI: 10.5958/0974-360X.2019.00957.0