INTRODUCTION:

The use of medicinal plant products as therapeutic agents and is manly involved or applied in the traditional type of medicine and especially seen in Ayurvedic and Unani treatment [1,2]. The major objective is to develop a mechanism related to quality assurance of these medicinal plant products used as drugs and applied in medical systems. One of the plant products i.e. pollen mainly reported in flowers and leaves [2-4]. These pollens mainly differ in size, shape and colour but it is considered as one of the most significant tools for identification of medicinal plant species [2-5]. In general, pollen contains protein, carbohydrates and reported amino acids (e.g. methionine, lysine, threonine, histidine, leucine, isoleucine, valine, phenylalanine, and tryptophan) and nucleic acid content as well.

The existence of vitamin is also reported in pollen e.g. fat-soluble vitamin (A, D and E; 0.1 %), and water-soluble (B1, B2, B6, and C; 0.6%) and acids (pantothenic, nicotinic and folic, biotin, rutin and inositol). In literature, pollen is also used by humans for various traditionally religious purposes and also use as supplementary food [6-8]. In contrast, pollen also used medically as well especially in case of bleeding stomach ulcers and some infectious diseases. This pollen may also be applied and used for the treatment and prevention of high-altitude-sickness syndrome. In some cases, most of the people are allergic to pollen, and reported specific allergens. Before using any pollen, sensitivities should be tested before pollen is applied/used for treatment or as a supplementary food [9,10].

The genus Calendula (Asteraceae) includes more than 25 herbaceous (annual or perennial) species, most common being Calendula officinalis and is widely reported in Dehradun, Uttarakhand region [11-14]. In Calendula officinalis especially flower used as a medicinal plant and showed various immunopharmacological properties i.e. antimicrobial, anti-inflammatory etc. In general, FDA (Food and drug administration) approved Calendula used as a spice and also applied in ointments, creams, oils etc. [11-14] Dianthus, annual flowering plant and reported only two species i.e. Dianthus superbus and Dianthus chinensis were identified [15, 16]. Both these species of dianthus reported number of chemical compounds or its derivatives e.g. secondary metabolites especially saponins and showed various immunopharmacological activities. To work on these two flowers and estimated its protein content and analysing its immunogenic effect (using typhoid vaccine) and also determining its antioxidant potential.

MATERIALS AND METHODS:

Collection of samples:

Pollen samples of flowers (Calendula officinalis and Dianthus chinensis) were collected in the month of January 2020 (total 4g of pollen in 2 days). Collected pollen was stored in air tight bags, and then stored at −80 °C pertaining to avoid any type of contamination.

Extraction of protein content from pollen:

For extraction of pollen protein from flowers (Calendula officinalis and Dianthus chinensis) using Tris-HCl (pH 7.2) and ice-cold acetone to remove fat constituents. In this study, pollen samples of flowers were weighed and crushed in mortar and pestle using dry ice to prepare fine powder and dissolved in Tris-HCl (pH 7.4) and then incubate it for 10 minutes at room temperature followed by centrifugation. Thereafter, acetone solution was added in the supernatant and incubated for 20 minutes. Finally, the extract was centrifuging, and supernatant was discarded and proteins settled at the bottom. The pellet was filter sterilized in autoclaved membrane filters (0.22µm) and determined the protein content using lowry method [17,18].

Standard and sample preparation:

Standard stock solution (1000µg/ml) of Bovine serum albumin, BSA and pollen protein from flowers (Calendula officinalis and Dianthus chinensis) was prepared in phosphate buffered saline, PBS. All solutions were stored at 5°C. The calibration curves of the standard (BSA) were prepared by serial dilution with PBS to yield 62.5–1000µg/ml. The calibration curves were drawn from the chromatograms as peak area versus concentration of BSA used as standard. In this study, pollen protein content was prepared in PBS and stored at low temperature.

During HPLC analysis, firstly all solutions (standards and pollen protein samples) were filtered through 0.20 µm PTFE syringe filter. In contrast, HPLC system consisted of a pump coupled to a variable UV absorbance detector (Model.1220 infinity LC, Aglient technologies, Mumbai) operated at 285nm. The chromatogram was recorded using an electronic integrator (Integrated diode array detector with spectra analysis). Chromatographic separation was carried out using a C18 reversed-phase (RP) analytical HPLC column (Agilent ZORBAX Eclipse plus C18 4.6 × 150 mm, 1.8μm (PN 959994-902), 245 bar). The mobile phase consisted of Acetonitrile–water (10:90, v/v). The flow rate was held constant at 0.6 ml/min.

Haemolytic activity:

This assay is done in whole human whole blood samples and consent letter was obtained from healthy volunteer. In this study, human whole blood was mixed with equal volume of sterilized PBS. This solution was centrifuged (4000rpm; 10 min) and collected the pelleted cells (containing RBCs). These pelleted cells was used for determining its haemolytic activity using different concentration of pollen protein (62.5-1000µg/ml) from Calendula officinalis and Dianthus chinensis. All these samples were incubated (37°C, 30 minutes) and centrifuged at 3000rpm for 10 min. Finally, supernatant was decanted and its haemoglobin content was estimated through spectrophotometer at 570nm [19].

Antigen specific immune assay:

In order to assess its immunological activity of pollen protein from Calendula officinalis and Dianthus chinensis using specific protein antigen (typhoid vaccine) in human lysed whole blood samples (10⁶ cells/ml, 100µl). Lysed human whole blood containing lymphocytes were cultured with pollen protein from flowers (Calendula officinalis and Dianthus chinensis) using variable concentration and treat with optimized concentration or dose of typhoid vaccine (25µg/ml; 10 µl) in phosphate buffered saline (PBS) containing heat-inactivated Fetal calf serum (FCS, 10%) along with penicillin and streptomycin (100μg/ml) in tissue culture 96 well plate for 24 h. After incubation, MTT dye (2.5 mg/ml; 10µl) was added and incubate the pollen samples for 2 h. Supernatant was discarded after centrifuging and observed formazan crystals settled at the bottom. Dissolve these formazan crystals using dimethyl sulphoxide (DMSO) solution and then analyzed its optical density (OD) at 570nm [20].

Antioxidant activity:

This assay was assessed through DPPH free radical assay using pollen protein from flowers (Calendula officinalis and Dianthus chinensis). In this assay, pollen proteins (two different sets) were reacted with DPPH radical in an ethanol solution. The reaction is mainly comprised of pollen protein (500µl), absolute ethanol (3000µl) and DPPH radical solution (300µl dissolved in 0.5mM ethanol). For these studies, DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced. The changes in colour (from deep violet to light yellow) were read [Absorbance (Abs)] at 517nm after 100 min of reaction using a UV-VIS spectrophotometer. For these studies, ethanol (3.3ml) and sample (0.5ml) selected as blank whereas control solution contained ethanol (3.5ml) and DPPH radical (0.3ml) solution. The scavenging activity percentage (AA%) was determined by using this equation i.e.

Antioxidant activity = 100 - [(Sample absorbance - Blank absorbance) x100/absorbance control] [21].

Statistical analysis:

The difference between control and treatment group of pollen protein from flowers is determined through one way ANOVA test (Bonferroni multiple comparison test).

RESULTS:

Protein content and HPLC analysis:

The results of these studies (using lowry method) showed the presence of protein content is higher in case of Dianthus chinesis (2.8mg/ml) followed by Calendula officinalis (0.78mg/ml) as compared to control. In addition, HPLC analysis was also performed and its peak revealed the presence of protein content with retention time (Fig.1).

HPLC results revealed the presence of a good percentage of protein content and these were detected at a wavelength of 270 nm due to sharpness of the peaks and proper baseline and recorded its retention time (Rt min), percent area and heights.

Haemolytic activity:

The effect of pollen protein extracted from Dianthus chinesis and Calendula officinalis for determining its haemolytic activity in human whole blood as shown in Fig. 2. These studies showed that pollen protein at higher concentration does not showed any haemolytic effect as compared to distilled water and control. Distilled water showed highest haemolytic activity as compared to control.

Fig. 2. Haemolytic activity in human whole blood.

Distilled water used as positive control for the study. After incubation, centrifuging the lysed human whole blood containing variable concentration of pollen protein and the supernatants were collect and estimated the free haemoglobin in the supernatants using spectrophotometer. The OD was measured at 405nm.

Antigen specific immune response:

The effect of pollen protein on lysed human whole blood using typhoid vaccine as shown in Fig. 3. The results of these studies showed that pollen protein at higher concentration showed decline in proliferation rate against optimized dose of typhoid vaccine antigen as compared to control. Overall, these studies reveal that pollen protein at higher concentration showed less immunogenic or declining in antigen specific T cell proliferation rate.

Fig. 3: Antigen specific proliferation assay.

Lysed human whole blood containing lymphocytes (10⁶ cells/ml, 100µl) were cultured with variable concentration of pollen protein (1.4 and 2.8mg/ml, 100 µl in Calendula officinalis; 0.39 and 0.78mg/ml, 100µl in Dianthus chinensis) and treat with optimized concentration or dose of typhoid vaccine (25µg/ml; 10 µl). All these details are mentioned in materials and methods section. The optical density (OD) was measured at 570nm. The difference between control and variable doses of pollen protein against optimized concentration of vaccine antigen is determined through one way ANOVA test (Bonferroni multiple comparison test). *P <0.05; **P < 0.01 and ***P < 0.001

Antioxidant activity:

The results of these studies showed that pollen protein extracted from Dianthus chinesis showed higher antioxidant activity (using DPPH) as compared to Calendula officinalis and control (Fig.4).

Fig. 4: Antioxidant assay using DPPH.

This assay was conducted to check the free radical scavenging ability of the pollen protein against DPPH free radical using fixed/optimized concentration of pollen protein and its values are expressed as Mean± S.E. The difference between control (DPPH) and variable doses of pollen protein is determined through one way ANOVA test (Bonferroni multiple comparison test). **P < 0.01 and ***P < 0.001

DISCUSSION:

Pollen collection from medicinal plant products especially flower and leaves reported various immunobiologically active substances. In literature, more than two hundred substances were identified and reported in pollen from different plant species [9,10]. Most of the valuable medicinal plants still reported are Peganum harmala, Psoralea corylifolia, Fumaria indica, Achillea millefolium, Acorus calamus, Arnebia nobilis, Origanum vulgare, Paeonia emodi, Rauwolfia serpentina were identified through palynological (pollen) studies [3-10]. These pollen based studies are routinely applied or used to analyse its metabolites in terms of its quality and safety for human health care. In general, most of the plant species containing pollen mainly protein, carbohydrates, phenolic compounds, lipids, amino acids, vitamins, minerals etc. The objective of our study is to determine the protein content in pollen of two different flowers and determine its antioxidant potential and also evaluating its immunogenic potential against typhoid vaccine in human whole blood and studied its interrelationships. This type of studies especially pharmacological were already conducted in rats and rabbits and showed that pollen confirmed its hypolipidemic activity (i.e. declining in the content of plasma total lipids and triacylglycerols) [6-9]. In addition, pollen is also recommended in acute and chronic inflammatory type of conditions, initial degenerative conditions, and cholestatic liver diseases as well [8-11]. In addition, nutritional properties of pollen and various metabolic processes are also reported and also recommended to administer pollen in the recovery period, after surgeries, and to people working hard physically and mentally. The beneficial effect of pollen is mainly reported in inflammatory type of conditions e.g. prostate gland [22]. In some studies, scientists confirmed that when pollen was administered along with some chemotherapeutic agents, the number of people who felt a significant therapeutic effect significantly increased. In this regard, we worked on pollen from two different flowers and determined its immunogenic potential and also determining its antioxidant activity.

Antigen specific immune based assay (using typhoid vaccine) is one of the most traditional type of protocol for determining its immunogenic potential of pollen protein. This study reveals that pollen protein at higher concentration showed declined in proliferation rate at higher doses as compared to control. In other words, pollen protein showed anti-inflammatory activity or less immunogenic and this activity could be declining in clonal expansion and proliferation of antigen specific T cells. In continuation of these studies, we focused on antioxidant potential of pollen protein from flowers through DPPH assay. This antioxidant assay is mainly considered them as one of the most valuable parameter pertaining to determining its profile through spectrophotometry method [21]. In general, DPPH assay (free radical method) is totally based on electron-transfer which ultimately produces a violet coloured solution in ethanol. So, free radical is readily stable at room temperature, which is reduced in the presence of an antioxidant molecule, giving rise to colourless ethanol solution. According to these studies, pollen proteins also showed antioxidant activity and its highest antioxidant activity is reported in protein of Dianthus chinesis as compared to Calendula officinalis. These results suggest that the antioxidant activity of pollen protein are exactly is in crude form and this activity is mainly influenced through various phenolic compounds which are used in the traditional medicine. Overall, pollen is more effective in terms of anti-inflammatory (declining in T cell proliferation) and also possess antioxidant property as well.

CONCLUSION:

This study revealed the presence of protein content in pollen and showing inhibition in antigen specific immune response but also showing antioxidant property as well. Further immunopharmacological investigations are required pertaining to find active components (primary or secondary metabolites) from flower pollen and to confirm the mechanism of action.

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Received on 03.05.2020 Modified on 02.07.2020

Research J. Pharm. and Tech. 2021; 14(6):2989-2994.

DOI: 10.52711/0974-360X.2021.00523