Uncovering the Phytochemical Profile, Antioxidant Potency, Anti-Inflammatory Effects, and Thrombolytic Activity in Dendrobium lindleyi Steud.

Background. Dendrobium genus has been used in traditional medicine to treat various illnesses. The study aims at examining the phytochemical, antioxidant, anti-inflammatory, and thrombolytic properties of the leaf, stem, and root of Dendrobium lindleyi Steud, and the relationship between phytochemicals and bioactivities is determined. Results. The qualitative screening found a variety of bioactive compounds, including alkaloids, coumarins, cardiac glycosides, glycosides, flavonoids, proteins, phenols, quinines, resins, steroids, saponins, tannins, and terpenoids, in varying amounts. The quantitative screening showed the highest concentration of alkaloids in the leaves (172.15 ± 1.22 mg/g), phenols in the root (203.55 ± 0.75 mg/g), flavonoids in the root (24.35 ± 0.42 mg/g), tannins in the leaves (105.06 ± 0.55 mg/g), and proteins in the root (194.12 ± 0.65 µg/ml). The root extract showed the highest antioxidant activity (IC50 = 58.24 µg/mL), the stem extract had the most increased thrombolytic activity (IC50 = 242.74 µg/mL), and the leaf extract had the most potent anti-inflammatory activity (IC50 = 61.79 µg/mL). Statistical analysis revealed a significant positive relationship (p = 0.05) between alkaloids (r = 0.96) and tannins (r = 0.9) with antioxidant, anti-inflammatory, and thrombolytic properties. Conclusion. The bioactivities of D. lindleyi, including antioxidant (root), thrombolytic (stem), and anti-inflammatory (leaf) activities, are linked to the phytochemicals detected in the screening.


Background
Te traditional medical systems of Ayurvedic, Siddha, and Unani have documented the use of orchids as medicine, as recorded by Charaka, Sushruta, and Vagbhata.With the growing demand for natural remedies, there is an increased interest in the medicinal use of orchids in traditional medicine [1].Te use of Dendrobium species as medicine has a long history, as it was mentioned in the Chinese pharmacopeia "Te Sang Nung Pen Tsao Ching" as early as 200 B.C. as a tonic, astringent, analgesic, and anti-infammatory substance [2].
Emperor Shen-Nung also described the medicinal benefts of Dendrobium in his "Materia medica."Modern research has confrmed the anti-infammatory, antifungal, antibacterial, and other pharmacological properties of Dendrobium species [3,4].Bangladesh has approximately 194 species of orchids, including 91 medicinal orchids [5][6][7][8][9].Dendrobium lindleyi Steud.It is one of the orchid species found in Bangladesh.Although its traditional use or therapeutic benefts are unknown, fresh and dried stems of D. lindleyi are used in traditional Chinese medicine to treat dehydration, fever, and eye health [10].Previous research has shown promising biological activity in the foral section of the plant [11]; however, the biological activity of the leaves, stem, and root of the plant still needs to be explored.Terefore, a comprehensive investigation of the phytochemical and pharmacological aspects of D. lindleyi is being conducted.Te study aims at validating its traditional uses and creating a foundation for further research towards developing new drugs from this species.

Plant Collection and Identifcation.
Plants were collected from Bandarban (Keokradong hill), Chittagong, in the southeast region of Bangladesh, in April 2019.Te area lies within latitude 22 °11′43″ North and longitude 92 °13′10″ East and runs along the border of Bangladesh.Te plant was identifed in the Chittagong University Herbarium (CTGUH), Department of Botany, University of Chittagong, where a voucher specimen (Accession no: KB_05/2019) was deposited for future reference.

Plant Extraction.
Fresh leaves, stems, and roots were harvested and thoroughly washed with water, room dried, and sun-dried.Ten, the samples were dried in an oven at 60 °for 72 hours.It was then ground into a coarse powder using a grinding machine and stored in an air-tight container for further investigation.25 gm of sample from each plant part were taken for further analysis.150 ml of methanol was added to each sample in a conical fask.It was then shaken well for 30 minutes, kept overnight, shaken again, and sonicated for 10 minutes.It was then fltered using the Whatman No. 1 flter paper.Te process was repeated thrice with methanol; then, the extract was evaporated below 51 °and dried.Te dried sample was kept as a crude sample for each part.

2.4.2.
Quantitative.Quantitative phytochemical tests were performed using the following standard procedures.
(1) Total Alkaloid Determination.For determining the total alkaloid content, fresh samples of leaves, roots, and stems (5 g each) were placed in 250 mL beakers and a mixture of 200 mL 20% acetic acid in ethanol was added.After 4 hours, the mixture was fltered and the extract was concentrated in a water bath to a quarter of its original volume.Concentrated ammonium hydroxide was gradually added until precipitation was complete.After settling, the precipitate was washed, fltered, dried, and weighed with the weight expressed as mg/g extract [15].
(2) Total Flavonoid Determination.For total favonoid estimation, standard solutions of quercetin were prepared and plant extracts were treated similarly.After incubation and spectral measurement at 510 nm, the favonoid content was expressed in mg quercetin equivalents (QE)/g of the dried plant extract [16,17].
(3) Total Phenol Determination.Methanol extracts (1 mg/ml) were analyzed to determine the total phenol concentration.A mixture of methanol extract, 10% Folin-Ciocalteu's reagent, and 7.5% sodium carbonate solution was prepared and incubated.Absorbance was measured at 760 nm using a UV spectrophotometer.Te total phenol concentration was determined from a calibration curve of gallic acid equivalents (GAE) and expressed as mg GAE/g of the extract [18,19].
(4) Total Tannin Determination.For total tannin determination, extracts (1 mg/ml) were mixed with water, Folin-Ciocalteu reagent, sodium carbonate solution, and distilled water.After incubation, absorbance at 700 nm was measured using a UV spectrophotometer.Tannin content was expressed in mg of tannic acid equivalents/g of the dried sample [20].
(5) Total Protein Determination.A phenolic complex with the maximum absorption at 660 nm was formed for total protein determination.Bovine serum albumin (BSA) was used as a standard protein.Samples were mixed with reagents and incubated.Ten, absorbance was measured at 660 nm using a UV spectrophotometer.Te protein content was expressed in mg/g of dried plant extract [21].assessed based on the free radical scavenging efect of the stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH, MWt.394.32) free radical activity with slight modifcation [22].Preparation of reagents of crude extract of the leaves, stems, and roots of D. lindleyi was conducted to prepare a range of concentrations (50, 100, 150, 200, and 250 µg/mL) in methanol.Ascorbic acid (Positive control) with similar concentrations was also prepared in methanol.0.004% DPPH solution was prepared in methanol.Te absorbance was measured at 517 nm using a UV-visible spectrophotometer.Te experiment was performed thrice.Te scavenging activity against DPPH was calculated using the following equation:

In Vitro Analysis
where A � Absorbance of DPPH solution (negative control) and B � Absorbance of DPPH solution (extracts/ascorbic acid).

Anti-Infammatory Activity.
Te anti-infammatory activities of the methanolic crude extracts of the leaf, stem, and root of D. lindleyi and the standard antiinfammatory agent acetyl salicylic acid were assessed based on inhibition of the albumin denaturation technique of the stable egg albumin denaturation inhibition activity with slight modifcation [23,24].Preparation of reagents of crude extracts of the leaf, stem, and root of D. lindleyi was carried out to prepare a range of concentrations (50, 100, 150, 200,250, and 300 µg/mL) in methanol and 1% aqueous solution of egg albumin, and the pH values (5.6 ± 0.2) of all mixtures were adjusted by 1N HCl.Acetylsalicylic acid with diferent concentrations (50, 100, 150, 200, 250, and 300 µg/ mL) was also prepared.Te absorbance was measured at 660 nm using a UV-visible spectrophotometer.Te experiment was performed thrice.Te anti-infammatory activity was calculated by using the following equation: where A � absorbance of egg albumin solution + methanol (negative control) and B � absorbance of egg albumin solution + extract/standard (positive control).

Trombolytic Activity.
A clot lysis experiment was carried out to check the thrombolytic properties of the plant extract.In this method [25], venous blood drawn from healthy volunteers are transferred in diferent preweighed sterile Eppendorf tubes (500 μL/tube) and incubated at 37 (3)

Statistical Analysis.
Finally, the study statistically examined total phytochemicals (alkaloids, phenols, tannins, favonoids, and proteins) for their efect on bioactivity (antioxidant, anti-infammatory, and thrombolytic).For this, regression analysis and Pearson's correlation coefcient analysis were performed by Microsoft Excel 2010.

Phytochemical Screening
3.1.1.Qualitative.Te responses of the plant extracts to various reagents provide valuable insights into their chemical composition and the presence of specifc alkaloid groups in diferent parts of the plant (Table 1).When tested with Dragendorf's reagent, the leaf extract displayed a "++" response, the stem extracted a "+++" reply, and the root extracted a "++" response.Similarly, when exposed to Hager's reagent, all parts of the plant-the leaf, stem, and root-demonstrated a "+++" response.Mayer's reagent produced distinct responses as well.Te leaf extract exhibited a "+++" response, the stem extracts a "+" reply, and the root extract a "+++" response.Furthermore, against Wagner's reagent, the leaf extract showcased a "+++" response, the stem extracts a "++" response, and the root extract a "++" reply.Lastly, the tannic acid reagent elicited "++," "+," and "-" responses from the leaf, stem, and root extracts.Moreover, coumarins, cardiac glycosides, favonoids, proteins, phenols, quinines, resins, steroids, saponins, tannins, and terpenoids were detected in varied amounts during the qualitative screening (Table 2).Te leaf, stem, and root reported traces of glycosides and saponins.Te resin was evident in the root but just a tiny amount in the stem and none in the leaf.(2) Total Flavonoid.In the present work, 13.86 ± 0.20, 1.63 ± 0.66, and 24.35 ± 0.42 mg•g −1 of favonoids were found in D. lindleyi in the leaf, stem, and root, respectively.Te highest amount of favonoid found in the root extract was 24.35 ± 0.42 mg/g.
(5) Total Tannin.Te total tannin contents of the leaf, stem, and root of D. lindleyi were found to be (105.06± 0.55, 53.12 ± 1.34, and 69.60 ± 1.21) mg•g −1 respectively.Te highest amount of tannin in the leaf extract was 105.06 ± 0.55 mg/g compared to the extracts of the stem and the root.

In Vitro Analysis
3.2.1.Antioxidant Activity.Te antioxidant activity was assessed using the DPPH free radical scavenging assay.Te antioxidant activity (Figure 2) of leaf, stem, and root extracts obtained IC 50 values of 111.79, 82.24, and 58.24 µg/mL, respectively.Compared to the standard (26.08 µg/mL), the root extract showed the most potent antioxidant activity, the stem extract displayed intermediate potency, and the leaf extract exhibited minor antioxidant potency among the tested plant parts (Figure 3).

Anti-Infammatory Activity.
Te anti-infammatory activity was evaluated through an albumin denaturation assay, which measures the ability of substances to prevent the denaturation (unfolding) of albumin, a protein.Te IC 50  4), with the leaf extract having the most potent anti-infammatory efect when compared to the standard (22.69 µg/mL).Te IC 50 values from the albumin denaturation assay measured the ability of the tested substances (standard, leaf, stem, and root extracts) to prevent the denaturation of albumin.A lower IC 50 value indicates more potent anti-infammatory activity.Te standard had the most powerful protective efect, followed by the root extract, the leaf extract, and the stem extract (Figure 5).Tese fndings give insights into the relative strengths of these substances in preserving the structural integrity of albumin.

Trombolytic Activity.
Te thrombolytic activity was measured using the blood clotting inhibition method.Te results of a thrombolytic activity assay conducted on various substances, including leaf extract, stem extract, and root extract, with IC 50 values of 275.06, 242.74, and 319.62 µg/mL, respectively, as compared to the standard (100 µL) (Figure 6).Te percentages of clot lysis and the IC 50 values provide insights into their efectiveness in promoting clot dissolution.Te thrombolytic activity was measured using the blood clotting inhibition method.Te results suggest that the standard and the tested extracts have varying levels of thrombolytic activity.Te stem extract showed the most potent thrombolytic activity, followed by the leaf extract.In contrast, the root extract exhibited the lowest thrombolytic activity (Figure 7).Te

Scientifca
IC 50 values indicate the concentration of each extract needed to achieve a 50% reduction in clot formation, with lower values suggesting more substantial thrombolytic efects.

Statistical Analysis.
In this analysis (Table 3), correlation values (r) were calculated as 0.96 for alkaloids, 0.9 for tannins, 0.7 for proteins, 0.51 for phenols, and 0.54 for favonoids which indicated a strong positive correlation for alkaloids as well as tannins, whereas they indicated moderate positive correlation for proteins, favonoids, and phenols.Statistical analysis showed that alkaloids (r � 0.96) and tannin (r � 0.9) have a substantial positive connection with antioxidant, anti-infammatory, and thrombolytic activities at a signifcance level of p < 0.05 signifcantly.

Discussion
In the phytochemical investigation, fve reagents tested alkaloids.When tested with Dragendorf's reagent, the leaf extract displayed a "++" response, the stem extracted a "+++" reply, and the root extracted a "++" response.Tis variation in responses suggests that the stem extract likely contains a higher concentration of alkaloids with secondary amine groups than the leaf and root extracts, which showed more moderate responses.Similarly, when exposed to Hager's reagent, all parts of the plant-the leaf, stem, and root-demonstrated a "+++" response.Tis collective strong response strongly indicates the presence of alkaloids containing methoxy or catechol groups in substantial quantities across the entire plant.Mayer's reagent produced distinct responses as well.Te leaf extract exhibited a "+++" response, the stem extracts a "+" reply, and the root extract a "+++" response.Here, the leaf and root extracts demonstrated a high response, suggesting the signifcant presence of alkaloids containing hydroxyl or amino groups.In contrast, the stem extract yielded a lower response to Mayer's reagent.Furthermore, against Wagner's reagent, the leaf extract showcased a "+++" response, the stem extract showcased a "++" response, and the root extract showcased a "++" reply.Notably, the leaf extract displayed the highest response, indicating the likely presence of alkaloids characterized by tertiary amines and phenolic hydroxyl groups.
While the stem and root extracts showed moderate responses, they target diferent alkaloid characteristics.Lastly, the tannic acid reagent elicited "++," "+," and "-" responses from the leaf, stem, and root extracts.Te leaf extract's "++" response suggests the likely presence of alkaloids that can react with tannic acid.Comparatively, the stem extract's lower "+" response suggests a lower quantity of these alkaloids, and the root extract's "-" response indicates an absence of reaction to this particular reagent.When the other phytochemical components of D. lindleyi were studied, a variety of bioactive compounds, including coumarins, cardiac glycosides, favonoids, proteins, phenols, quinines, resins, steroids, saponins, tannins, and terpenoids, were discovered.Te highest concentration of alkaloids was found in the leaves, while the root contained the highest levels of phenols and favonoids.Traces of glycosides and saponins were found in the leaves, stems, and roots, and resin was only present in the root.Tese fndings are consistent with the previous studies [26][27][28][29][30]. Te presence of bioactive phytochemicals in D. lindleyi suggests that it may have a wide range of therapeutic properties [31].
Te antioxidant activity was assessed using a free radical scavenging assay.An IC 50 value of 111.79 µg/mL was obtained for the leaf extract, indicating that it has comparatively lower antioxidant potency than the stem and root extracts.Tis means that a higher concentration of the leaf extract is required to achieve the same level of antioxidant protection as the other extracts.In contrast, the stem extract demonstrated greater antioxidant potency than the leaf extract.Its IC 50 value was 82.24 µg/mL, suggesting that it can achieve a 50% reduction in oxidative damage at a lower concentration than the leaf extract.Te most robust antioxidant activity was observed in the root extract, which exhibited the lowest IC 50 value of 58.24 µg/mL among the three extracts.Tis signifes that the root extract possesses the highest antioxidant potency.Te antioxidant efects are thought to be due to favonoids, saponins, and phenolic substances [32,33].
Te anti-infammatory efcacy was determined using the heat-induced albumin denaturation assay.Te standard substance demonstrated an IC 50 value of 22.69 µg/mL.Tis means a standard substance concentration of 22.69 µg/mL efectively prevents albumin denaturation by 50%.Te lower the IC 50 value, the more potent the substance is in preserving the native structure of albumin.Te leaf extract had an IC 50 value of 61.79 µg/mL.At a 61.79 µg/mL concentration, the leaf extract can protect albumin from denaturation by 50%.Te stem extract showed an IC 50 value of 112.11 µg/mL.At a 112.11 µg/mL concentration, the stem extract can inhibit albumin denaturation by 50%.Te root extract displayed an  6 Scientifca IC 50 value of 70.76 µg/mL.At a 70.76 µg/mL concentration, the root extract can protect albumin from denaturation by 50%.Tis value positions the root extract as relatively more efective in preserving the native structure of albumin than the stem extract but still less efective than the standard.Te anti-infammatory properties may be due to saponins, terpenoids, alkaloids, glycosides, and tannins [34,35].Te thrombolytic activity was measured using the blood clotting inhibition assay.Te standard exhibited a clot lysis percentage of 52.1%.Tis means that the standard, when used at a volume of 100 µL, could cause a 52.1% dissolution of the blood clot.Te standard likely represents a positive control with known thrombolytic activity.Te leaf extract showed a range of clot lysis percentages between 30.2% and 47.1%.Tis suggests that the leaf extract has varying degrees of thrombolytic activity within this range.Te IC 50 value for the leaf extract was measured as 275.06 µg/mL.Tis IC 50 value indicates that the leaf extract achieves a 50% reduction in clot formation at a concentration of 275.06 µg/mL.Te stem extract also displayed a range of clot lysis percentages, ranging from 31.8% to 49.6%.Te IC 50 value for the stem extract was 242.74 µg/mL.Tis value indicates that the stem extract efectively reduces clot formation by 50% at a 242.74 µg/mL concentration.Te root extract demonstrated a range of clot lysis percentages from 9.6% to 39.8%.Te IC 50 value for the root extract was 319.62 µg/mL.Tis indicates that the root extract achieves a 50% reduction in clot formation at a concentration of 319.62 µg/mL.Te thrombolytic activity may be due to alkaloids and tannins [36,37].Tus, the thrombolytic, anti-infammatory, and antioxidant properties of D. lindleyi may be due to its alkaloids, phenolic compounds, and other phytocompounds [38].Statistical analysis also indicates a signifcant positive correlation between the antioxidant, anti-infammatory, and thrombolytic activities with the content of alkaloids and tannins [39,40].

Conclusion
In summary, the results indicate that the diferent parts of D. lindleyi (root, stem, and leaf) have varying levels of efectiveness in their antioxidant, thrombolytic, and antiinfammatory activities.Te root extract is the most efective as an antioxidant, the stem extract has the highest thrombolytic activity, and the leaf extract is the most potent in antiinfammatory activity.Tese fndings can have implications for potential applications of these plant extracts in health and medicine.However, further research is usually necessary to fully understand their mechanisms of action and potential benefts.

Limitations of the Study.
Te research on D. lindleyi, a high-altitude plant with no recorded medicinal use, aimed at examining its phytochemical properties and evaluating its potential as an antioxidant, anti-infammatory, and thrombolytic agent during the COVID-19 pandemic.However, the study was limited by the need for in vivo testing, which could not be conducted due to time constraints.

2. 5 . 1 .
Antioxidant Activity.Te antioxidant activities of the methanolic crude extracts of the leaves, stems, and roots of D. lindleyi and the standard antioxidant ascorbic acid were 2 Scientifca °Cfor 45 minutes.After clot formation, the serum is completely removed (aspirated out without disturbing the clot formed).Each tube having a clot is again weighed to determine the clot weight.Each Eppendorf tube containing the clot is labelled correctly, and 100 μL of plant extract is added to the tubes.All the tubes are then incubated at 37 °C for 90 minutes and observed for clot lysis.After incubation, the fuid obtained is removed and tubes are again weighed to keep the diference in weight after clot disruption.Streptokinase and water are positive and negative (nonthrombolytic) controls, respectively.Te experiment is repeated three times with the blood samples of diferent volunteers.Te diference obtained in weight taken before and after clot lysis is expressed as a percentage of clot lysis as follows: clot weight � weight of clot − containing tube − the weight of tube alone, % of clot lysis � weight of released clot clot weight   × 100.

Table 1 :
Qualitative test for alkaloids.

Table 3 :
Results of regression correlation analysis of the total content in bioassays.