Exploring The Therapeutic Potential of Sea Buckthorn Enriched Polyherbal Oil Formulation for Alleviating Diabetic Neuropathy Pain

Diabetes mellitus is a heterogeneous chronic metabolic disorder characterized by high blood glucose levels due to abnormalities in insulin secretion and/or action. Carbohydrate, fat, and protein metabolism dysfunctions due to hyperglycaemia are systemic and impair the functions of many organs in the body. These perturbations are progressive and result primarily from the deleterious effects of hyperglycaemia and its associated metabolic derangements on the normal architecture and physiology of these complications comprise organ damage, dysfunction, and finally organ failure and involve some body organs, especially eyes, kidneys, heart, and nerves. It manifests as retinopathy with progression to blindness. Nephropathy and possible renal failure; Complications associated with the kidney. These include coronary heart disease and hypertension. Complications related to the nerves are manifestations like neuropathies which can be peripheral and/or autonomic. Diabetes, the rapidly growing global epidemic, has become one of the most urgent and significant health problems facing the human society currently urbanization and the shift towards modern lifestyle habits have been widespread with high rates of economic development which paralleled the rising prevalence of diabetes around the world. [1]

1.1 Emerging risks of Diabetes mellitus

Complications classically ascribed to diabetes mellitus include macrovascular disorders such as coronary heart disease, stroke and peripheral arterial disease, and microvascular disorders that entail diabetic kidney disease, retinopathy and peripheral neuropathy. Moreover, heart failure is the most common first manifestation of cardiovascular disease in individuals with type 2 diabetes mellitus. In some countries or regions, cancer is the leading cause of death in individuals with diabetes mellitus. Diabetes mellitus presents with traditional complications such as stroke, Coronary heart disease and heart failure, peripheral neuropathy, retinopathy, diabetic kidney disease and peripheral vascular disease Despite advances in the management of diabetes mellitus, Associations between diabetes mellitus and cancer, infections, functional and cognitive disability, Liver disease and affective disorders are instead becoming prevalent.

1.2 Diabetic Neuropathy

Diabetic neuropathy is a common and serious complication of diabetes mellitus, affecting up to 50% of patients with type 1 or type 2 diabetes. It is a neurodegenerative disorder that primarily impacts the peripheral nervous system, often starting in the distal sensory neurons and progressing proximally. Key risk factors include poor glycaemic control, longer diabetes duration, older age, obesity, hypertension, dyslipidemia, and smoking. The two main types are distal symmetric polyneuropathy and autonomic neuropathy. Early detection and intervention are critical, as diabetic neuropathy significantly contributes to morbidity, mortality, and non-traumatic amputations. Effective management relies on optimized blood sugar control, multifactorial interventions, and symptomatic treatment. [2]

1.3 Types of Diabetic neuropathy

• Peripheral neuropathy: This type of neuropathy in most clinical practice is seen as sensory-neuronal neuropathy in patients with diabetes. It is defined by slowly progressive symmetric numbness in the distal foot, paraesthesia, with or without neuropathic pain, and absent Achilles tendon reflexes. It may or may not be related to weakness. This neuropathy comprises the main underlying cause of hospital admissions for foot ulcers, infection, and gangrene.

• Proximal Neuropathy: Proximal neuropathy is a diabetes-related nerve disorder causing pain and weakness in the hips, thighs, or legs. A subtype, diabetic amyotrophy, presents with sudden, one-sided pain and muscle weakness.

• Autonomic Neuropathy: Poor glycaemic control in diabetes can damage autonomic nerves, causing issues like irregular heart rate, digestive problems, erectile dysfunction, dry skin, and sweating disorders. This may lead to ulcers, gangrene, and potential limb amputation.

• Focal Neuropathy: Focal neuropathy in diabetes is a sudden, painful nerve injury due to reduced blood flow, often affecting cranial and peripheral nerves. It usually resolves in about six weeks and includes conditions like Bell’s palsy and carpal tunnel syndrome. [3]

1.4 Pathophysiology of Diabetic Neuropathy

The exact mechanism of diabetic neuropathy is not known but it is taken up that toxic effect of hyperglycaemia is considered as the cause of development of diabetic neuropathy. Other factors include:

• Enhanced Polyol Pathway Activity:

Hyperglycaemia from reduced insulin increases polyol pathway activity, where aldose reductase converts glucose to sorbitol, consuming NADPH. Sorbitol then converts to fructose, raising NADH levels. Excess sorbitol causes intracellular osmotic stress, contributing to diabetic complications.

• Oxidative Stress:

Oxidative stress is initiated by the formation of harmful free radicals due to glucose metabolism and deficient of antioxidant defence which plays a major role in the pathogenesis of diabetic neuropathy.

• Microvascular Changes:

Diabetic neuropathy is caused by reduced blood flow to nerves due to damaged small blood vessels, leading to poor oxygen supply, swelling, and pressure buildup. High blood sugar worsens this by further limiting oxygen to sensory nerves. Despite increased leg blood flow in some cases, oxygen deficiency remains a key factor, possibly linked to sympathetic nerve dysfunction.

• Damaged Nerves:

Damaged nerve endings in diabetic neuropathy may generate abnormal signals interpreted as pain by the CNS. Nerve injury also alters ion channel expression, increasing nerve excitability and contributing to neuropathic pain. [4]

1.5 WHO Data on Diabetic Neuropathy

According to the World Health Organization (WHO), the global number of individuals with diabetic neuropathy has more than tripled since 1990, reaching 206 million cases in 2021. It is estimated that 50% to 66% of people with diabetes will eventually develop diabetic peripheral neuropathy (DPN) during their lifetime.

The prevalence of DPN increases with both age and the duration of diabetes. Studies indicate that after 5 years of living with diabetes, approximately 26% of patients develop peripheral neuropathy. This figure rises to 41% after 10 years of the condition.

Mortality rate of Diabetes:

According to the World Health Organization (WHO), diabetes mellitus was responsible for approximately 26.9 deaths per 100,000 population in India in 2021. This means that diabetes was the direct cause of around 1.6 million deaths that year. The diabetes mortality rate increased 3% by age from 2000 to 2019.

Impact of Diabetic Neuropathy on Mortality:

A study revealed that adults with diabetes who also have peripheral neuropathy and a foot ulcer face more than twice the risk of mortality compared to those without these complications. [5]

1. 6. Statement of Problem

Diabetic neuropathy is a chronic and progressive complication of diabetes that significantly impairs the quality of life due to persistent nerve pain, inflammation, and oxidative stress. Existing pharmaceutical treatments often provide limited relief and are associated with side effects such as sedation, gastrointestinal issues, or dependency. There is a growing demand for safer, natural alternatives that offer effective symptom management with minimal side effects. Despite the therapeutic potential of medicinal plants like Sea Buckthorn, Ashwagandha, and Curcumin, their synergistic use in a topical polyherbal oil formulation for diabetic neuropathy remains underexplored. This project aims to develop and evaluate a stable, effective, and skin-compatible herbal oil enriched with Sea Buckthorn and other neuroprotective botanicals to provide a holistic, side-effect-free approach to managing diabetic neuropathy pain.

2. PRE-FORMULATION STUDIES

2.1 Phytochemical Profile:

SEA BUCKTHORN SEED OIL

Appearance: Clear, viscous, oily

Colour: Golden yellow to Amber

Odour: Characteristic nutty to mildly pungent

pH: 2.5 (acidic)

Viscosity: 40-50 mPa’s at 25°C

Refractive index: 1.465-1.475

Solubility: Soluble in fixed oils and alcohol

Phytochemical Constituents

• Fatty acids: Linoleic acid (omega 6), Alpha linoleic acid (omega 3), oleic acid (omega 9), Palmitoleic acid (omega 7), Saturated fatty acids

• Flavonoids: Isorhamnetin, kaempferol, Quercetin, Proanthocyanidine, Catechin

• Vitamins: Vitamin C, Vitamin E (Tocopherols and Tocotrienols), Vitamin A (Carotenoids like beta-carotene), Vitamin K, B-complex vitamins – B1, B2, B6, folic acid

• Phenolic Compounds: Gallic acid, Caffeic acid, Ferulic acid, Ellagic acid

• Phytosterols: Beta-sitosterol, Campesterol, Stigmasterol

• Triterpenoid, Saponins, Tannins, Alkaloids, Amino acids and minerals. [6]

Therapeutic benefits

Sea buckthorn seed oil, rich in omega-3, omega-6, vitamin E, and phytosterols, offers anti-inflammatory, antioxidant, and neuroprotective benefits. It helps relieve diabetic neuropathy symptoms by reducing nerve inflammation, aiding regeneration, and improving circulation. Additionally, it promotes wound healing, skin repair, heart and liver health, immunity, and acts as a natural moisturizer. [7]

ASHWAGANDHA OIL

Appearance: Clear to slightly turbid viscous fluid

Colour: Light brown to reddish brown

Odour:  Characteristic earthy, slightly pungent herbal smell

Texture: Oily, Smooth, and Non-sticky after application

pH: 5.5-6.5

Viscosity: 130-159cP (Depends on temperature)

Refractive index: 1.47 – 1.48

Solubility: Insoluble in water, miscible with oils & ethanols

Phytochemical Constituents: Withanolides, Alkaloids, Saponins, Flavonoids, Steroidal lactones, Tannins, Fatty acids, Fatty acids, Vitamin E (Tocopherols). [8]

Therapeutic Benefits:

• Ashwagandha oil possesses neuroprotective, anti-inflammatory, analgesic, and antioxidant properties, making it effective for managing diabetic neuropathy.
• It lowers nerve inflammation, promotes neuron regeneration, alleviates neuropathic pain, such as burning and tingling, and improves local circulation.
• It also boosts stress resilience, protects against oxidative damage, and nourishes the skin, all of which help to prevent diabetic foot problems when applied topically. [9]

MAHANARAYAN OIL

Appearance: viscous, clear to slightly turbid oily liquid

Colour: Reddish brown to dark brown

Odour: Strong herbal, slightly pungent and spicy aroma

Texture: Smooth and oily, leaves a greasy layer when applied

pH: 5.5-6.5 (in emulsion form)

Viscosity: 120-150cP at 25°C

Refractive index: 1.470-1.480

Solubility: Insoluble in water, miscible with oils and ethanol

Phytochemical Constituents: Alkaloids (Piperine, Berberine), Flavonoids (Quercetin, Kaempferol), Tannins (Polyphenolic compounds), Glycosides (Iridoid and Cardiac Glycosides), Terpenoids & Volatile oils (Cineole, Camphene, Menthol), Saponins (Withanosides, Shatavarin), Steroids (Plant Steroids- Beta-sitosterol). [10]

Therapeutic Benefits:

• Mahanarayan oil is a traditional Ayurvedic preparation that contains powerful anti-inflammatory, analgesic, antioxidant, and neuroprotective effects. 
• In diabetic neuropathy, it reduces nerve inflammation, relieves burning and tingling sensations, promotes nerve regeneration, improves peripheral circulation, and relaxes muscular stiffness. 
• It is also beneficial in treating joint pain, arthritis, muscular spasms, sciatica, and general body aches. 
• When applied topically, its nourishing and renewing ingredients improve skin health while also strengthening the musculoskeletal and nervous systems. [11]

CURCUMIN EXTRACT

Appearance: Fine crystalline or amorphous powder (dried extract)

Colour: Bright yellow to orange

Odour: Mild, Characteristic earthy or spicy odour

Texture: Smooth, Fine powder

pH (suspension): Slightly acidic (around 6.5)

Partition coefficient: Log P ~ 3.3 (Lipophilic)

Melting point: ~183°C

Solubility: Practically insoluble in water, Soluble in ethanol & Oils.

Stability: Sensitive to light, heat, and alkaline pH

Appearance in Base: Disperses well in oils with mild heating

Phytochemical Constituents: Curcumin (Principle active compound), Demethocycurcumin, Bisdemethoxycurcumin, zingiberene (mild extract form).[12]

Therapeutic Benefits:

• Applied topically in oil formulations, curcumin extract has strong anti-inflammatory, antioxidant, and analgesic properties.
• Reducing oxidative stress in nerve tissues, lowering inflammation in peripheral nerves, and calming burning, tingling, or numbness helps to ease the discomfort related with diabetic neuropathy.
• Since it also improves microcirculation, promotes nerve repair, and shields nerve cells from more damage, curcumin is a great treatment for neuropathic pain.
• Its mild skin-care qualities also help to reduce the irritation, redness, and localized edema brought on by ongoing diabetic nerve pain. [13]

CAMPHOR

Appearance: Crystalline, waxy solid

Colour: White or colourless

Odour: Strong, penetrating, aromatic

Texture: Brittle, Dry crystals

Partition coefficient: Log P ~2.4 (Lipophilic)

Melting point: ~175-177°C

Boiling point: ~204°C

Solubility: Soluble in ethanol, oils and chloroform, Insoluble in water 

Volatility: High (volatile at room temperature)

Stability: Light and heat sensitive

Phytochemical Constituents: Terpenoid (Monoterpene ketone), Monoterpenoids. [14]

Therapeutic Benefits:

• Camphor is an effective counter irritant, causing a cold sensation that helps divert from pain.
• In diabetic neuropathy, it improves local blood circulation, lowers nerve discomfort, and alleviates burning, tingling, and numbing.
• It’s analgesic, anti-inflammatory, or antispasmodic properties provide rapid relief from neuropathic pain.
• It also relieves muscle spasms, softens tight tissues, and promotes total neuromuscular calmness, making it an important element in external oil preparations for nerve pain therapy. [15]

PEPPERMINT OIL

Appearance: Clear, mobile liquid

Colour: Colourless to pale yellow

Odour: Strong, fresh, sharp, Menthol like aroma

Texture: Watery, less viscous

pH: 3.0-6.0

Refractive index: 1.460-1.467

Solubility: Soluble in alcohol, oils, and chloroform, Insoluble in water

Stability: Light sensitive, should be stored in airtight amber bottle

Phytochemical Constituents: Terpenoids (Menthol, Menthone), Esters (Menthyl acetate), Flavonoids, Oxides (cineole-eucalyptol). [16]

Therapeutic Benefits:

• Peppermint oil has potent analgesic, and antispasmodic properties.
• Its main ingredient, menthol, produces a cooling feeling which helps stop pain signals and alleviates burn or tingling feelings that result from diabetic neuropathy.
• It improves local circulation, reduces muscle tension, and alleviates nerve irritation and neuropathic pain.
• Additionally, its calming and vasodilatory qualities help to relieve stiffness and promote relaxation of afflicted nerve tissues. [17]

3. AIM & OBJECTIVES

3.1 AIM:

To develop and evaluate a Sea Buckthorn-Enriched Polyherbal Oil as a natural, safe, and effective alternative for managing diabetic neuropathy pain, focusing on its antioxidant properties while minimizing side effects.

3.2 OBJECTIVE:

• To develop a stable polyherbal oil formulation for alleviating diabetic neuropathy pain.

• To understand the mechanism of action and safety profile of the formulation compared to existing treatments.

• To develop a cheaper formulation for alleviating diabetic neuropathy pain.

• To contribute to the development of a natural, alternative remedy for managing diabetic neuropathy pain, offering a safer option than conventional treatments.

4. EXPERIMENTAL WORK

4.1 Materials and Methods

Material Used in the Formulation

Table 1. Ingredients Used and Their Brand Name

FORMULATION OF POLYHERBAL OIL

Table 2. Formulation Table

1. Assemble the required apparatus.
2. Accurately weigh the required quantities of Sea Buckthorn oil, Ashwagandha and Mahanarayan oil in separate beakers.
3. In a water bath, warm these three oils gently on a low flame and do not over heat.
4. Mix the Sea Buckthorn oil, Ashwagandha oil and Mahanarayan oil together after heating.
5. Now, accurately weigh the required quantity of Camphor.
6. Add Camphor in the above mixture before it gets cooled and stir it well using a glass stirrer until it completely dissolves in the oil.
7. To the above mixture add the Curcumin extract in accurate quantity.                                                                                                         
8. At last, add the peppermint oil and stir thoroughly.                                        
9. Store the formulated oil in amber coloured glass bottle and label them.                                                                                                               

5. EVALUATION OF POLYHERBAL OIL

Characterization of Formulated Oil

The characterization study includes a methodical technique used to assess and comprehend the key features for a pharmaceutical and herbal composition. It entails examining the biological, chemical, and physical characteristics in order to guarantee that the formulation is high quality, stable, beneficial, and safe to use. This study is an important aspect of product development since it provides scientific data to justify the formulation's intended use.

5.1 PHYTOCHEMICAL TESTS

Phytochemical investigation or phytochemical tests is the qualitative and/or quantitative examination of formulations for the presence of bioactive components that includes alkaloids, flavonoids, tannins, saponins, glycosides, terpenoids and or phenolic compounds.  These tests aid in determining the potential for treatment and medicinal properties in the polyherbal oil formulations.

1. Identification Tests for Sea Buckthorn:

1. DETECTION OF ALKALOIDS

Mayer’s Test:

1 mL of the extract is combined with 1 mL of Mayer’s reagent. The presence of alkaloid is confirmed by appearance of white or yellowish precipitate.

Wagner’s Test:

To 1 mL of extract, few drops of wagners’s reagent is added. The confirmation of Reddish-brown precipitate indicates the presence of alkaloids.

2. DETECTION OF FLAVONOIDS

Shinoda Test:

Small piece of magnesium ribbon is added to 2 mL of the extract. Carefully add 2–3 drops of concentrated HCl. The red color change is due to the presence of flavonoids.

Alkaline Test:

To 2 mL of the extract, few drops of 10% NaOH solution is added. Then, a few drops of dilute HCl is added to the solution. Intense yellow color appears on addition of NaOH and disappears after adding HCl leaving a light yellow colour indicating the presence of flavonoids.

3. DETECTION OF PHENOL

Ferric Chloride Test:

To 1 mL of the extract of oil formulation, few drops of 5% ferric chloride solution is added. A different blue, green, purple, or black coloration signifies the presence of phenolic constituents.

4. DETECTION OF TANNINS

Lead Acetate Test:

To 1 mL of the extract of oil formulation, added a few drops of 10% lead acetate solution. The presence of tannins is confirmed by white or yellow precipitate.

5. DETECTION OF CARBOHYDRATES

Molisch’s Test:

To 1 mL of the extract of oil formulation, 2-3 drops of Molisch’s reagent is added, followed by 1 mL of concentrated H?SO? along the sides of the test tube without stirring. Formation of a violet or purple ring at the junction indicates the presence of carbohydrates.

Benedict’s Test:

1 mL of the extract is combined with 1 mL of Benedict’s reagent. Boil the mixture for 2–5 minutes in a water bath. Confirmation of orange/red, or brick-red precipitate describes the presence of reducing sugars.

2. Identification Tests for Other Phytoconstituents:

1. DETECTION OF STEROIDS AND TRITERPENOIDS

Salkowski Test:

1 mL of the extract is dissolved in 1 mL of chloroform. Carefully add 2 mL of concentrated H?SO? along the side of the test tube to form a layer. Presence of a reddish-brown ring at the interface shows the presence of steroids and triterpenoids.

2. DETECTION OF CURCUMIN

Boric Acid Test:

Small quantity of boric acid is mixed into a few milliliters of ethanol containing the extract. Formation of a reddish or orange-red complex indicates the presence of curcumin, due to complex formation between curcumin and boric acid.

3. DETECTION OF CAMPHOR

2, 4-Dinitrophenylhydrazine (DNPH) Test:

A few drops of 2,4-DNPH reagent is added to the oil extract. The appearance of a yellow, orange, or red precipitate indicates the presence of a carbonyl functional group, suggesting compounds such as camphor or acetone.

4. DETECTION OF PEPPERMINT OIL

Test for menthol – Nitric Acid Test:

To the extract 1 mL of concentrated nitric acid is added, followed by gentle heating. Formation of yellow to orange coloration or yellow nitration product indicates the presence of menthol. [18]

5.2 EVALUATION TESTS

The prepared oil Formulation was subjected to various evaluation tests such as physical characterization which includes colour, odour & appearance. Additionally, various physico-chemical parameters such as pH, viscosity, spreadability and other parameters were analysed to ensure the formulation's stability, compatibility, and effectiveness for topical application.

1. Organoleptic Parameters:

• Colour: The oil Formulation in a glass beaker was placed against a white background under natural day light and colour was observed.

• Odour: Take a few drops of oil in a clean porcelain dish or on blotting paper. Bring it close to the nose (without inhaling deeply). The type, intensity, and pleasantness of the smell was assessed (e.g., herbal, pungent, floral, mild).

• Texture: 2-3 drops of the oil was applied on the back of a clean hand. It was spread gently using the fingertip in circular motions; and the texture was noted (greasy, smooth, thick, thin, sticky, or silky).

• Clarity: The oil was poured into a clean, dry test tube and was hold against a light source or white background. Then it was observed for clarity, cloudiness, suspended matter, or sediments.

• Homogeneity: The oil bottle was shaken gently and visually inspected for any phase separation, sedimentation, or layering.

• After Feel: The oil was applied in small amount on the skin (hand or forearm). It was rubbed gently and allowed to absorb. After 5–10 minutes, any sensation: greasy, dry, smooth, sticky, hydrated, cooling or any irritation was noted.

2. Physico-Chemical Properties:

1. Determination of pH

The pH meter was calibrated using standard buffer solution (pH 7.0). The electrode was washed with distilled water and then gently dried with a tissue paper. The pH electrode was then dipped in the oil formulation and a stable reading was recorded.

2. Determination of Viscosity

The viscosity of the oil formulation was determined using a Brookfield Viscometer. The oil was placed in a clean beaker and maintained at 25 ± 1°C. A suitable spindle (Spindle No. 3) was selected and immersed into the oil without touching the sides or bottom. The viscometer was operated at 10 rpm, and the viscosity reading was recorded in centipoise (cP) after stabilization.

3. Determination of Specific Gravity

A pycnometer (specific gravity bottle) is used to measure the specific gravity of the oil-based formulation. The empty, clean, and dry bottle was first weighed and its weight noted (W1). It was subsequently filled with the oil formulation and reweighed (W2). After cleaning and drying, the bottle was filled with distilled water and weighed (W3). The specific gravity was calculated using the formula for specific gravity and the result was expressed as a unitless ratio compared to water.

SG = Weight of the oil (g) / Weight of an equal volume of water (g)

4. Determination of Spreadability

The spreadability of the oil formulation was evaluated using two glass slides and a standard weight method. One ml of the oil was placed between two clean glass slides, which were then pressed uniformly by placing a 50g weight on top for 5 minutes. After removing the weight, the diameter of the spread oil film was measured using a ruler. The spreadability (S) of the formulation was calculated using the formula:

S = M (g) × L (cm) / T (sec)

5. Determination of Washability

To assess washability, about 1 mL of the oil formulation was applied to a small section of the skin. The oil was gently rubbed and left undisturbed for 5 minutes. The area was then rinsed under running tap water for 30 seconds without using soap or detergent. The extent to which the oil was removed was visually assessed and categorized as easily washable, moderately washable, or poorly washable.

6. Determination of Irritancy/Sensitivity

The irritancy potential of the oil formulation was evaluated by applying a small amount (approximately 0.5 mL) of the oil to a marked area (1 cm²) on the forearm of healthy human volunteer. The site was observed for any signs of redness, itching, swelling, or rashes at regular intervals—specifically at 30 minutes, 1 hour, and 24 hours after application. Volunteers were instructed not to wash the area during the observation period. No adverse reactions indicated that the formulation was non-irritant and safe for topical use. [19]

5.3 STABILITY STUDIES

Stability studies are conducted to determine how well a pharmaceutical or cosmetic product maintains its physical, chemical, microbiological, and functional properties over time under the influence of various environmental factors such as temperature, humidity, and light.

1. Accelerated Stability Test

The oil formulation was stored in airtight glass containers at 37°C ± 2°C for a period of three months. Observations were recorded at three intervals—after 1 month, after 2 months, and after 3 months. At each interval, the formulation was evaluated for any changes in color, odor, pH, and viscosity. The results were compared with initial baseline values to assess the formulation’s physical and chemical stability under accelerated conditions.

2. Thermal Stability Test

The thermal stability of the oil formulation was evaluated at the same three intervals -after 1, 2, and 3 months. At each time point, approximately the stored oil was subjected to three cycles of heating at 45–60°C followed by cooling to room temperature. After each cycle, the sample was inspected for phase separation, precipitation, or any visible changes. The absence of such changes indicated thermal stability over time. [20]

Formulations for Stability Study

6. ANTIOXIDANT ACTIVITY

Antioxidant activity is the capacity of a compound to neutralize free radicals—unstable molecules that can damage cells, proteins, and DNA. This activity helps protect the body from oxidative stress, which is linked to aging and diseases like cancer, diabetes, and heart disorders. ntioxidants help neutralize free radicals by donating electrons, thereby protecting cells from potential damage.

6.1 DETERMINATION OF TOTAL PHENOLIC CONTENT

Determination of Total Phenolic Content (TPC) is a method to measure the amount of phenolic compounds in a sample, which are key natural antioxidants. It’s commonly done using the Folin–Ciocalteu reagent (FCR), which reacts with phenolics to produce a blue color. The color intensity, as determined using a spectrophotometer, indicates the concentration of phenolic compounds. Since phenolic compounds can neutralize free radicals, higher TPC indicates stronger antioxidant activity. [21]

PROCEDURE:

1. Preparation of Standard Gallic Acid Solution:

1. Weigh accurately 10 mg (0.010 g) of gallic acid using an analytical balance.
2. Dissolve in a 100 mL volumetric flask using distilled water.
3. Mix well until the gallic acid is completely dissolved.
4. This forms a 100 µg/mL (100 ppm) stock solution.
5. Preparation of Working Standard Solutions:

From the 100 µg/mL stock solution, pipette out the required volumes and dilute to 10 mL using distilled water.

Table 3. Preparation of Stock Solution

3. Steps for Dilution:

1. Take a clean 10 mL volumetric flask for each concentration.
2. Accurately pipette the required volume of the stock solution (100 µg/mL) into each flask.
3. Dilute up to the mark with distilled water and shake well to mix.
4. Label the prepared solutions appropriately.

4. Preparation of Standard Curve (Gallic Acid Calibration Curve):

1. Pipette 1 mL of each prepared gallic acid standard solution (10, 25, 50, 75 µg/mL) into separate test tubes.
2. Add 5 mL of Folin–Ciocalteu reagent to each tube and mix well.
3. After 5 minutes, add 4 mL of 7% sodium carbonate (Na?CO?) solution to each tube.
4. Mix thoroughly once more and incubate the tubes in a water bath for 30 minutes.
5. Record the absorbance at 760 nm using a UV-Vis spectrophotometer.
6. Plot the calibration curve: Absorbance vs. Concentration of gallic acid (µg/mL).

5. Extraction of Sample:

1. Dissolve 2 mL of oil in 10 mL methanol to prepare the stock solution.
2. Take 1 mL from this stock and dilute it to 10 mL with methanol.

6. Preparation of Sample:

1. Pipette 1 mL of each sample solution into separate test tubes.
2. Add 5 mL of Folin–Ciocalteu reagent and mix well.
3. After 5 minutes, add 4 mL of 7% sodium carbonate (Na?CO?) solution to each tube.
4. Mix properly and incubate in a water bath for 30 minutes.
5. Record the absorbance at 760 nm using a UV-Vis spectrophotometer.
6. Use the gallic acid standard curve to estimate the total phenolic content in the sample.

7. Preparation of Blank:

Instead of gallic acid or sample, take 1 mL of methanol and follow the same procedure. [22]

Standard, Sample and Blank Solution

Two formulations of the polyherbal oil (F1 and F2) were successfully developed using varying concentrations of the active herbal ingredients. Both formulations were evaluated for their physical appearance, physicochemical properties, and stability.

Labelling of the Polyherbal Oil

The physical properties of the oil formulations were evaluated, and the findings are presented below:

Table 4

The various physicochemical parameters of the oil formulations (F1 and F2) were evaluated to assess their physical and chemical properties. The results are mentioned below:

Table 5

• The pH ranged between 5.1 and 5.3, which is ideal for skin application (typical safe skin pH range: 4.5-5.5).
• The viscosity values ranged from 175 to 189 cP, suitable for easy spreadability and absorption (Acceptable range: 100-300 cP).
• Specific gravity values were within expected limits (0.85-0.95 g/mL) for oil-based formulations.
• F1 showed slightly better spreadability (8 g·cm/sec) than F2 (7.5 g·cm/sec), enhancing ease of application.
• Both formulations were easily washable and non-irritating, indicating good safety and user acceptability.

Both formulations (F1 and F2) exhibited acceptable physicochemical characteristics. F1 showed slightly better spreadability and lower viscosity compared to F2, while both were easily washable and caused no skin irritation, indicating good topical compatibility.

7.3 Stability Study

The stability study of the oil formulation included Accelerated stability testing and thermal stability testing at 3 intervals in 3 months and different parameters were assessed. The results are mentioned below:

7.4 Phytochemical Tests

The phytochemical examination of the polyherbal oil formulation confirmed the presence of alkaloids, flavonoids, phenols, tannins, steroids, terpenoids, and other chemical constituents. These bioactive compounds suggest that the formulation possesses significant antioxidant and anti-inflammatory activity, which are essential for the treatment of diabetic neuropathy pain. A summary of the results is provided in the following table.

Table 7. Identification Tests for Sea Buckthorn

Table 8. Identification Tests for Other Phytochemicals