Introduction
Since prehistoric times, medicinal plants have been discovered and used in ayurvedic and traditional medicine practice. Medicinal plants contain numerous chemical compound which acts as a preventive, curative, and protective agent against various disease. One of the medicinal plants used for memory enhancement is Celastrus paniculatus (CP) (Fig. 1). Genus Celastrus has more than 100 species of shrubs, which are distributed over China, Japan, Tropical Asia, North America, and Australia. In India, four species are confined, of which CP willd. CP is also called Jyothishmati or Malkangani in Ayurveda and Sanskrit and is commonly called as “Black seed oil plant” (Table 1) [1].
CP is used in ancient traditional medical treatment for various ailments. In Ayurveda, more importance is given to natural plants, which possess to have many medicinal values without any side effects. Like that, CP is known for its biological activities and stands as a useful drug in the treatment of rheumatism, dyspepsia, gout, and insomnia [2,3] it and remarkable role in cognitive enhancement, nootropic activity, antioxidant activity, anti-inflammatory activity and it is a one of its kind to improve the functions of nervous system, increases the nervous resistance, nourishes the neuronal cell lines and also it possesses anti-convulsant, anti-aging, tranquilizing effect, anti-anxiety and anti-depressant activity. CP is used in many ailments and central nervous system disorders such as epilepsy, fainting, dental treatments, skin disease, asthma, wound treatment, and a few more useful benefits to list down. Due to all these remarkable benefits, CP has been used for ages. CP contains fatty acids such as oleic acid, palmitic acid, linoleic acid, linolenic acid, and stearic acid. It contains tannins, and sugars but no starch [4]. Phytochemical contents of CP include Oil, alkaloids, polyalcohol, triterpenoids, sterols, celapagin, celapanin, celapanigin, celastrine, paniculatine and polyesters [5]. Beta-amyrin, beta-sitosterol, paraffinic hydrocarbons, and pentacyclic triterpene were isolated from a nonsaponifiable fraction of the CP seed oil [6].
Figure 1.
Celastrus paniculatus.
Figure 2.
Pictorial representation of methodology.
Many studies have been conducted to prove its efficacy to treat neuronal disorders. CP is included in the marketed formulation as one of the contents for memory-associated issues. Many chemical and synthetic molecules were synthesized and studied for memory-related issues but due to their side effects on other organs, no satisfactory results are remarked to treat the disease. Hence, exploring alternative ways to treat this disease through traditional or natural ways is much needed and it is commendable that natural herbs with medicinal values show better results more effectively. Also, when compared to modern medicine, medicinal plants are cheaper and cost-effective. To rule out memory-related problems, many compounds are tested for their efficacy. Likewise, phospholipids were studied to treat neuro-related issues as these phospholipids are involved in the synthesis of acetylcholine, a neurotransmitter involved in muscle control, circadian rhythm, memory, and other neuronal functions. Eukaryotic cell membranes are composed of phospholipids, a specialized group of lipids performing a variety of functions; they are glycerophospholipid, phosphatidylcholine (lecithin), phosphatidylethanolamines (cephalin), phosphatidylserine & phosphatidylinositol.
There is a lack in the study of CP seed oil for its minor and major constituents. Some studies related to triacylglycerol and fatty acid profiles have come up among them polar lipids, fat soluble vitamins, and sterols made an important point. In this present study, seeds of CP were selected and analyzed for lipid classes and fatty acid composition. The objective of this experiment is to analyze CP seed for its physicochemical, pharmacognostical, and fatty acid composition and estimation of important phospholipids such as phosphatidylcholine and phosphatidylethanolamine. The study goal is to obtain an informative profile about the chemical nature of CP oil. The results, furthermore will remarked as a potential nutraceutical and economical utilization of CP seeds as a new source of edible oils to treat dementia, Alzheimer’s disease, and Parkinson’s disease.
Materials and Methods
CP seeds were provided as a gift sample from JSS Ayurveda Medical College and Hospital, Mysuru, Karnataka.
Standards used for phospholipids identification, phosphatidylcholine, and phosphatidylethanolamine from soybeans were purchased from Sigma Aldrich. Chemicals used for estimation were available in the laboratory.
Pre extraction
CP seeds were subjected to powder using a mixer grinder and stored in an airtight container for further studies. Seed powder was used to conduct experiments for its pharmacognostical and physicochemical evaluation (Fig. 2).
Determination of moisture content
Firstly, seed powder was analyzed for its moisture content. To determine moisture content, loss on drying of the seed sample needs to be evaluated. The mass, loss on drying is measured as a percentage of weight per weight. Water and volatile materials in the raw drugs are both identified by the test for loss on drying. Crude drugs inevitably contain moisture, which must be removed as much as possible. A porcelain dish was filled with 1.5 g of carefully weighed seed powder. For 2 hours, the sample was kept in the oven at 105ºC. It was then brought to room temperature in a desiccator, and the process was repeated until a steady weight was noted. The following calculation was used to compute the percentage of loss due to drying [7].
Determination of ash value
A crude extract’s quality and purity can be assessed using its ash value, especially when it is in powdered form. When burned, crude pharmaceuticals typically leave an ash that is composed of sodium, potassium, calcium, and magnesium silicates, carbonates, and phosphates. Particularly in situations when silica may be present or when the drug’s calcium oxalate concentration is extremely high, a greater limit of acid-insoluble ash is applied [8].
Determination of total ash value
Two grams of the powdered medication, precisely weighed, were placed in a silicon crucible before being ignited and measured. The powder was then burned in a muffle furnace by progressively raising the heat until it reached temperatures no higher than 450ºC for 4 hours until it was carbon-free. The crucible was weighed after cooling. Using the following formula, the percentage of ash was determined in relation to the air-dried drug.
Determination of water-soluble ash value
With 25 ml of water, the ash was heated for 10 minutes. It was filtered using “ashless” filter paper, and any residue was then heated through twice. Filter paper and residue were combined and gently heated in the crucible until no more vapors were released. After cooling in the desiccator, it was weighed. Using the following formula, the proportion of water-soluble ash was determined in relation to the air-dried.
Determination of acid insoluble ash value
Ash was heated for 10 minutes with 25 ml of dilute hydrochloric acid. It was filtered using “ashless” filter paper and any residue was rinsed twice in hot water. Filter paper and residue were combined and gently heated in the crucible until no more vapors were released. After cooling in the desiccator, it was weighed. Using the following formula, the percentage of acid-insoluble ash was determined in relation to the air-dried drug [8].
Total lipid extraction
The seed powder was extracted with a mixture of chloroform and methanol in a 2:1 (vol/vol) ratio. The mixture was magnetically stirred for about 4 hours and filtered to separate the seed material. The filtrate was washed in water and the organic phase was separated, and it was dried on anhydrous sodium sulphate. The solvent was evaporated to obtain the lipid extract (oil) which was stored in the refrigerator until further analysis [9].
Determination of acid value
Principle
A small quantity of free acid is usually present in oils and fats along with triglycerides. The free acid content is known as acid value and it increases during storage due to rancidity and by the action of air, moisture, and microorganisms. They cause hydrolysis of ester-link and oxidation of double bonds of triglycerides forming low molecular weight free fatty acids which develops a disagreeable or offensive odor to fats and oils. Thus, makes it unsuitable for consumption or medicinal use. The amount of acids present in most fixed oils is generally low and acid value is used to eliminate low-grade and rancid oil.
Estimation of free fatty acid in lipid samples is based on alkali metric titrations. The official method involves the dissolving of the sample in an equal volume of a mixture of previously utilized ethanol and ether and free fatty acids are determined by titrating against standard alkali (0.1 MKOH) using phenolphthalein as an indicator till a pink color persists [7].
Procedure
Standardization of 0.1 M KOH.
Accurately weighed 630 mg of oxalic acid in a 100 ml volumetric flask and added sufficient water to dissolve and finally make up the volume. Pipetted 10 ml of standard oxalic acid solution into a 250 ml conical flask and added 1–2 drops of phenolphthalein solution as an indicator. Titrated against 0.1 M 1 KOH until the pink colour was obtained. Noted the burette reading. Repeated till concordant readings were obtained.
Assay.
10 g of oil sample, weighed precisely, was dissolved in 50 ml of an equal volume mixture of ethanol and ether, which had previously been used with 0.1 M KOH. 1 ml of phenolphthalein solution was added, and the amount was adjusted against 0.1 M KOH until the solution remained just barely pink after 30 seconds of shaking. The acid value was calculated from the formula
where a=volume in ml of 0.1 N KOH consumed for sample titrations
W=weight in g of the oil sample solution
Determination of saponification (SAP) value
Principle
The amount of potassium hydroxide needed to neutralize the free fatty acids and saponified esters contained in 1 g of sample or material is known as the SAP value. It indicates the number of mg of KOH required to neutralize free acids resulting from hydrolysis of 1 g of oil of fat. In the official determination, the sample is subjected to alkaline hydrolysis followed by SAP by boiling with a known quantity of alkali in excess, and excess alkali is determined by titrating against standard acids using phenolphthalein as an indicator. A blank titration is carried out and the difference in volume (blank titre value-sample titre value) indicates the amount of alkali used for SAP. Since each molecule of fat requires three molecules of KOH to saponify it, it is evident that SAP not really indicate a number of fat molecules per gram of fat. The SAP number becomes a measure of the size of the fat molecule, a high SAP value denotes fatty acids with low molecular weights, and vice versa [7].
Table 1.
Scientific classification and vernacular names of CP [10].
| Scientific names | Regional names |
|---|---|
| Kingdom: Plantae Phylum: Tracheophytes Class: Magnoliopsida Order: Celastrales Family: Celastraceae Genus: Celastrus Species: C. Paniculatus |
Bengali: Kijri, Malkangani English- Black oil plant, climbing staff plant, intellect plant Gujarati- Malkangana Hindi- Malkangani Kannada- Kariganne, Kougilu, Jotishmati Bhavamga Konkani- Malkangoni Malayalam- Cherupunna, Jyothishmathi, Killithinipanji, Paluzhavam, Valuzhavam, Marathi- Kanguni, Malkangoni Oriya- Korsana, Pengu Sanskrit- Alavan, Kangu, Jyotishmati, Tamil- Kuvarikuntal, Mannai-K-Katli, Valuluvai Telugu- Kasara-tige, Maneru Urdu- Malkanguni |
Procedure
Standardization of 0.5 M HCl.
Accurately weighed 2.65 g of anhydrous sodium carbonate and dissolved in 100 ml volumetric flask with water and made up the volume to 100 ml. Pipetted 10 ml of standard sodium carbonate solution into a 250 ml conical flask and added 2 drops of methyl orange indicator and titrated against 0.5 M HCL acid until a faint red colour persists. Titration was repeated to obtain concordant volume.
Assay.
Two grams of the oil to be tested were precisely weighed and added to a 200 ml flask with a reflex condenser. Added 25 ml of 0.5 M alcoholic KOH, accurately measured, and boiled under reflex in a water bath for 30 minutes. Rotating the contents often to allow them to cool, adding 1 ml of phenolphthalein solution, and titrating it against 0.5 M HCL acid (a ml). Repeated the procedure again but left out the drug being tested i.e., blank (b ml). The value of SAP was calculated from the following formula.
Ester value
The ester value is the quantity of potassium hydroxide needed to saponify 1.0 g of the substance’s esters in mg. The difference between the SAP and the acid value represents the Ester value
Ester value=SAP value – Acid value
Refractive index
The ratio of the sin of the angle of incidence to the sin of the angle of refraction of a light beam traveling from air into the substance is known as the refractive index (n) of that substance with respect to air. It changes depending on the wavelength of the light used to measure it.
Unless otherwise stated in the specific monograph, the refractive index, n20D determined at 20° ± 0.5° in relation to the sodium D-line’s wavelength (λ=589.3 nm). Due to the fact that temperature considerable affects the refractive index, the temperature needs to be carefully regulated and maintained. For the majority of the refractive index measurements, the Abbe refractometer is practical, but alternative refractometers with similar or higher precision may be employed [7]. First, turn on the light and calibrate the instrument using water. Adjust the refractometer scale knob to get a clear interface between the illuminated and dark regions. Note down the index of refraction using a telescope scale. Repeated thrice to get the concurrent value.
Viscosity
The temperature of the substance being examined must be precisely regulated since even tiny temperature variations can result in noticeable changes in viscosity. The temperature should be maintained to within ± 0.1° for usual pharmaceutical purposes.
Spindle viscometer
The spindle viscometer rotates a spindle that is submerged in the liquid to measure viscosity. Utilizing conversion factors from the scale reading at a specific rotational speed, one can directly derive relative values of viscosity (or apparent viscosity) [7]. To measure viscosity, the spindle was cleaned and then attached to the instrument. The spindle should rotate in the oil sample until a constant reading is displaced on the viscometer. Repeat in the same way three times and have to find out the average value
Density
A substance’s density, or more specifically, its volumetric mass density, is its mass per unit volume. Density is most frequently represented by the symbol; however Latin letter D may also be used. Density is mathematically, defined as mass divided by volume [7]. Measure the volume of the oil in millilitres and weigh the oil in grams. Now divide the mass in grams by the volume in millilitres to arrive at the density of oil. Repeat three times in the same way.
Column chromatography to determine the composition of lipid classes in extracted lipid sample
Column chromatography was performed following a reported protocol. By using silica gel column chromatography, the extracted lipids were segregated into neutral lipids, glycolipids, and phospholipids. The sample was loaded on to silica gel and was successively eluted with chloroform to obtain neutral lipid, acetone to obtain glycolipid, and methanol to obtain the phospholipid fractions [11,12].
Gas chromatography (GC) to determine the fatty acid composition of extracted lipid
Fatty acid composition by GC: GC was used to identify the lipids’ fatty acid content. According to published research, the lipid sample was transformed into fatty acid methyl esters (FAME) using the methanol-sulphuric acid (2% v/v) reagent. An Agilent 6,890 gas chromatograph connected to a flame ionization detector outfitted with a DB 225 capillary column (30 m × 0.25 mm × 0.25 µm) (J and W scientific, USA) was used to conduct the GC analysis of the FAME. The schedule for the column temperature was 2 minutes at 160ºC, 5 minutes at 230ºC, and 20 minutes at 230ºC. The split ratio was 10:1, with the injector temperature being 230ºC. N2 was used as the carrier gas, with a flow rate of 1 ml/minute. The flow rates of hydrogen and air were 300 and 30 ml/minute, respectively, and the detector temperature was 270ºC. By comparing the retention durations with a combination of standard FAMEs, C4-C24 (Supelco, USA), the fatty acids were identified. Every FAME sample was examined twice, and average results are shown [13].
Results
Discussion
The extracted seed oil of CP was brown in color, and the yield was about 50.16%. Physicochemical analysis of the seeds of CP shows loss on drying 3% w/w, total ash value 11%, water soluble ash value 0.5% w/w, acid insoluble ash value 4.95% w/w. The density of the oil is 0.89, the acid value is 1.78, SAP value is 284.32, which gives the ester value as 282.54. The refractive index is 1.472 and the viscosity is 50.15. All the values are tabulated in Table 2. By column chromatographic method, lipid classes were estimated. It was found to contain more neutral lipids (93.5%) than glycolipids (65%). Other research investigations about the phospholipids in CP oil were reported but unfortunately, we could not get the fractions of phospholipids in the current study protocol. The exact reason for not getting these fractions by this method could not be explained, but future studies may be done to estimate these phospholipids using a method developed by Donata et al. [14]. The composition of the lipid classes is tabulated in Table 3.
Table 4 reports the fatty acid composition of the oil. Nine fatty acids were detected in the extracted seed oil of CP, ranging from C14:0 to C20:1. Among the fatty acids, the predominant constituent was found to be oleic acid (C18:1) and palmitic acid (16:0) followed by linolenic acid (18:3) and linoleic acid (18:2). In this oil, oleic acid (35.30%), linolenic acid (16.22%), linoleic acid (15.90%), were identified as the main unsaturated fatty acids, while palmitoleic acid (0.14%) and eicosenoic acid (0.11%) were present in a lesser amount. Similarly, palmitic acid (27.39%) is the main saturated fatty acid with stearic acid (3.93%), myristic acid (0.61%), and arachidic acid (0.37%) in a smaller amount. Fatty acid composition in this current study is similar to those found in earlier studies done by Ramadan et al. [16], and Sengupta and Bhargava [17]. However, a study by Sengupta and Bhargava [17] and Sengupta et al. [18], the report found to be palmitic acid (31.26% and 32.8%) as the major fatty acid, but the current study was found to contain oleic acid (35.30%) to be the major fatty acid which is similar to study done by Ramadan [16] and Rana et al. [19]. Remarkably, arachidic acid and eicosenoic acid were found to be present in the current study, which was not estimated in the previous research studies. The current study proved that the composition of its fatty acid composition was similar to the oils which are edible and also if we can remove the odor of the seed oil by purification method, this CP oil could be considered as one of the sources of edible oil. Compared to the yield of the other edible and nonedible oils like Glycine max (20%), Linum usitatissimum (43%), Benincasa hispida (18.9%), Helianthus annus seed (35%), Jatropha curcas (40%), Vernicia fordii (18%), CP (50.16%) has highest oil content. CP oil has higher amounts of unsaturated fatty acids than saturated fatty acids.
Table 2.
Pharmacognostical and physicochemical results.
| Experiment name | CP | Codex alimentarus standard [15] |
|---|---|---|
| Total yield (%) | 50.16% | - |
| Color | Brown | Characteristic |
| Density | 0.89 (g/ml) ± 0.015 | 1.0 |
| Acid value | 1.78 ± 0.07 | 4.0 KOH/g oil |
| SAP value | 284.32 ± 0.29 | 248–265 |
| Ester value | 282.54 ± 0.07 | |
| Refractive index | 1.472 ± 0.004 | 1.4677–1.4705 |
| Viscosity | 50.15 ± 0.25 | 920–1,000 kg/m3 |
| Moisture content (%) | 3 ± 0.57 | 10% |
| Total ash value (%) | 11 ± 0.57 | 12 |
| Water soluble ash value (%) | 0.5 ± 0.05 | 1.5 |
| Acid insoluble ash value (%) | 4.95 ± 0.02 | 0.5 |
Table 3.
Composition of lipid classes in the seed oil.
| Neutral lipids (%) | Glycolipids (%) | Phospholipids (%) | |
|---|---|---|---|
| CP | 93.5 | 6.5 | 0 |
Table 4.
Fatty acid composition neutral lipids of the seed oil.
| Fatty acid compound | Amount (%) |
|---|---|
| Myristic acid (14:0) | 0.61 |
| Palmitic acid (16:0) | 27.39 |
| Palmitoleic acid (16:1) | 0.14 |
| Stearic acid (18:0) | 3.93 |
| Oleic acid (18:1) | 35.30 |
| Linoleic acid (18:2) | 15.90 |
| Linolenic acid (18:3 ALA) | 16.22 |
| Arachidic acid (20:0) | 0.37 |
| Eicosenoic acid (20:1) | 0.11 |
Acknowledgment
The authors acknowledge DST- Karnataka Science and Technology Promotion Society (KSTePs), Government of Karnataka for the financial support.
Conflict of interest
The authors declare that they have no conflict of interest.
