Evaluation of In Vitro Antimicrobial, Cytotoxic, Thrombolytic, and Antiarthritic Property of Different Parts of Bari Orchid

Many different herbal extracts have historically been utilized to treat microbe-induced infections, injuries, cancer, thrombosis, and arthritis. The purpose of this study was to determine the antibacterial, cytotoxic, in vitro thrombolytic, and in vitro antiarthritic properties of ethanolic extracts of stem and seed of Bari orchid 1 (BO) plant. This orchid plant was developed by the Bangladesh Agriculture Research Institute (BARI) in Gazipur. Fourteen microbes were employed in the antimicrobial investigation, and samples of orchids were compared to ciprofloxacin as a reference. The BO/seed extract was found to possess more antibacterial activity. The lethality test of brine shrimps was used to assess the LC50 values. The BO/stem extract exhibited a higher cytotoxicity potential, in comparison to the BO/seed extract. Two concentrations (1000 and 100 ppm) and two incubation times (24 hours and 1.5 hours) were used to assess the thrombolytic activity of the extracts. Regarding the thrombolytic effect, the BO/stem extract has demonstrated greater promise. Furthermore, the herbal extract's antiarthritic activity was investigated at four different concentrations, and the results were evaluated in comparison with those of diclofenac sodium. When comparing BO/stem extract to other extracts, the greatest values for protein denaturation were obtained.


Introduction
Since the beginning of time, herbal medicines have been used to treat a wide range of illnesses [1].From ancient times, these medicines were formulated by the therapeutic expertise of generations of physicians who had been practicing for centuries [2].Nature has given us a gift in the form of medicinal plants that can be used to treat and prevent diseases [3].Orchids are attractive fowers that have high value in society as ornamental plants.In addition, orchids are also known for their usage, especially in traditional medicine [4].
Bari orchid 1 (BO) is an orchid that possesses a long fower stalk with 12 to 15 forets, the petal's outer side is creamy white, and the petal's inner color is reddish-brown with a distinct maroon color tip and a golden yellow lip (Figure 1).Te fower has a sweet fragrance.Tis orchid is developed in Bangladesh Agriculture Research Institute (BARI)situated in Jaintapur, in the Sylhet district of Bangladesh [5].So far, no conventional applications have been established.Tus, traditional applications have no efect on the phytochemical and pharmacological properties.
Antimicrobial resistance is increasing globally (particularly in developing countries) as a result of the overuse and misuse of antibiotics [6].As a result, antibiotic resistance causes 700, 000 to several million deaths per year [7,8].Te World Health Organization says that the threat of antimicrobial resistance needs to be fxed as soon as possible [9].Natural antimicrobial compounds that can treat microbial infections well have been found in a lot of medicinal plants [10].Medicinal plants would be the best source to acquire various drugs, as declared by the World Health Organization [11].However, there are some limitations to old and newly introduced antimicrobial agents, e.g., short life expectancy, higher side efects, etc. [12,13].Since resistant clinical isolates are quickly spreading around the world, it is most important to fnd new antimicrobial agents.
Trombosis (blood clot) is a critical medical condition that is usually found in the veins of the arms, pelvis, thigh, lower leg, etc.To retrieve from this leads to disability, illness, and even death [14].Cardiovascular disease is the leading global cause of death, accounting for more than 17.9 million deaths in 2015, with a projected annual death rate of more than 23.6 million by 2030 [15].Te most common examples of thrombolytic agents are tissue plasminogen activator, streptokinase, urokinase, antistreptokinase, etc. Tese main agents dissolve the clots [16].Trombolytic agents do not work without adverse efects, such as bleeding, and to work best, a large quantity of fbrin-specifc medicines are required [17].Signifcant eforts have been made to discover and develop thrombolytic, antithrombotic, antiplatelet, and anticoagulant agents from natural constituents, e.g., various animal and plant sources, which will open new horizons to encourage the advancement of proper alternative thrombolytic therapy [18].
One of the autoimmune diseases, arthritis, is recognized by the co-occurrence of pain, stifness, and swelling.It is brought on by synovial joint infammation as a result of an immune-mediated reaction.Around the world, arthritis afects one in fve elderly people [19].NSAIDs, sulfasalazine, D-penicillamine, cyclophosphamide, cyclosporine, methotrexate, azathioprine, glucocorticoids, anakinra, etanercept, abatacept, and infiximab, among others, have been used to treat arthritis [20][21][22].But there are some risks, such as gastrointestinal ulcers, stomatitis, problems with the heart and blood vessels, pulmonary toxicity, myelosuppression, hematologic nephrotoxicity, hepatic fbrosis, cirrhosis, diarrhea, and local reactions at the injection site [23].Because of this, it is very important to make new antiarthritis medicines from medicinal plants that are cheap and have few side efects.
Secondary metabolites such as alkaloids, saponins, terpenoids, tannins, favonoids, glycosides, inulin, steroids, terpenoids, phlorotannin, phenols, essential oils, resins, and naphthoquinone give plants their strong pharmacological properties.As treatments for diseases, these substances work by changing the way the body works [24,25].Because people don't know enough about safe dosages and because some plants have toxic byproducts, using local plants as a source of medicine may have some bad efects [26].
In the current study, the in vitro antimicrobial, thrombolytic, antiarthritic, and cytotoxicity efect of these orchid plant extracts were evaluated because there is no scientifc evidence of the antimicrobial, thrombolytic, antiarthritic, and cytotoxicity potential of BO extracts.

Preparation of Extract.
BO was collected from the Horticulture Division, Bangladesh Agriculture Research Institute, Gazipur, Bangladesh.Te plants were washed with ethanol and dried separately at room temperature (22 ± 0.5 °C) for 15 days and grounded to powder form.Ten, 250 g of ground plant parts were soaked in 1.0 L ethanol for seven days using screw-capped reagent bottles.Filtrates were obtained through the Buchner funnel by Whatman flter paper No. 11 and concentrated by rotary evaporator (operating below 40 °C and lower pressure).Diferent plant extracts were then suspended in distilled water and mixed vigorously by a vortex mixer to make the desired concentration.

Test for Alkaloids.
Mayer's Test: 0.2 g of each extract was taken in test tubes and boiled on a steam bath after adding 5 mL of 2% HCl.Ten, the sections were fltered.After that, 1 mL of fltrates was taken in separate test tubes, and two drops of Mayer's reagent were added.Precipitation with a creamy white color indicates the presence of alkaloids.

2.2.2.
Test for Flavonoids.0.2 g of each extract and 10 mL ethyl acetate were taken in test tubes.Ten, the test tubes were heated at 100 °C for 3 minutes using a water bath.After  Evidence-Based Complementary and Alternative Medicine that, each extract was fltered, and fltrates were drawn for the below tests: (1) Ammonium test: 4 mL of the fltrates were shaken vigorously with 1 mL of 1% diluted ammonia.Ten, the layers were allowed to separate.Te yellow coloration of an ammonia layer proves the presence of favonoids.-colored(2) Aluminum chloride test: 4 mL of each fltrate was shaken vigorously with 1 mL of 1% aluminum chloride and allowed to sit for precipitation.Te formation of yellow-colored precipitation justifes the presence of favonoids.Ten, those were shaken vigorously and fltered.To the fltrates, fve drops of Molisch reagent were added and shaken vigorously again.After that, 1 mL of fuming sulfuric acid was carefully added to the test tubes.Te appearance of a brown ring at the interface indicates the presence of carbohydrates.

Test for Proteins
(1) Biuret's Test: 3 mL of extracts, 1 mL of 4% w/v sodium hydroxide, and 1 mL of 1% w/v copper sulfate were taken in test tubes.Te change in color of the extracts from blue to violet or pink proves the presence of proteins.(2) Xanthoproteic Test: 3 mL of each extract and 1 mL of concentrated sulfuric acid were taken in test tubes.Firstly, white precipitate forms, which then turns yellow on boiling; orange precipitation forms after the addition of 1 mL ammonium hydroxide, confrming the presence of proteins, e.g., tyrosine and tryptophan.

Antimicrobial Screening.
Te disc difusion method (CLSI guideline) was used for screening the antibacterial and antifungal properties of plant extracts as described by Daoud et al. [30,31] test bacteria and fungi.Ten those were kept in a refrigerator at 4 °C for about 24 h.Te upside of petri dishes was placed on the bottom to allow for adequate difusion of materials from the discs to the surrounding agar medium.Ten, each petri dish was inverted and placed in an incubator at 37 °C for 24 hours.Teir activity was measured by the diameter of the zone of inhibition, which was given in millimeters [32,33].Tis showed how well they stopped microorganisms from growing.
2.4.Brine Shrimp Lethality Bioassay.Te brine shrimp lethality bioassay was used to predict that the ethanolic extract of BO would be cytotoxic [34,35].For the experiment, 1 mg of the extracts were dissolved in dimethylsulfoxide (DMSO) and solutions of varying concentrations (100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.19, and 0.09 mg/mL) were obtained by the serial dilution technique using simulated seawater.Te solutions were then poured in the pre-labeled test tubes, which contained ten live brine shrimp nauplii in 5 mL simulated seawater.Te test tubes were examined with a 3× magnifying glass, and the number of brine shrimp nauplii that survived after 24 hours was counted.Te absence of controlled forward motion during 30 seconds of observation was defned as mortality [36].Te percentage of lethality was calculated for each extract and control from this data.Vincristine sulfate and DMSO were used as the positive and negative controls, respectively.Te values of LC 50 were found by plotting the logarithm of concentration against the percentage of deaths in Microsoft Excel Plus 2016 [37].After 24 hours, the number of brine shrimp nauplii that were still alive was counted, and the following equation was used to fgure out the percentage of deaths [34,38]: Percent of mortality � no. of dead nauplii after 24 h initial no. of live nauplii   × 100.
(1) 2.9.Phosphate Bufer Saline pH 6.4.In a 1000 mL volumetric fask, 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, and 800 mL distilled water were taken and mixed correctly.Ten, the pH of the solution was adjusted to 6.4 using 1 N HCl and flled with the needed amount of distilled water, making up the mark of 1000 mL.

2.10
. Procedure.To evaluate the antiarthritic activity, 2.8 ml of phosphate-bufered saline (pH 6.4), 2 ml of various plant extract concentrations (66.5, 125, 250, and 500 ppm), and 0.2 ml of fresh hen's egg albumin were taken in each test tube. 2 mL of distilled water was served as control. 2 mL of varying concentrations of diclofenac sodium (66.5, 125, 250, 500 ppm) were served as the standard.After that, the mixtures were incubated at 37 ± 2 °C in a BOD incubator for 15 minutes.Ten, the mixtures were heated at 70 °C for 5 minutes in a water bath.After cooling the solutions, absorbance was measured at 660 nm by a UV-Vis spectrophotometer.As a control, phosphate-bufered saline was used.Te percent inhibition of denaturation of proteins was calculated using the following formula: where Vt = absorbance of test sample and V °= absorbance of control.
Te tests were done in triplicate.

Preliminary Phytochemical Screening.
For the qualitative determination of the phytochemicals of ethanolic extract of the diferent parts of Bari orchid 1, these extracts were subjected to preliminary phytochemical screening according to standard procedures.Te phytochemicals discovered are listed in Table 1.Te positive signs of the phytochemicals confrm the presence of saponins, steroids, glycosides, terpenoids, carbohydrates, phenols, favonoids, tannin, alkaloids, and proteins in the plant extracts (Table 1).

UV-Visible Spectrophotometric Analysis.
Te identifcation of phytoconstituents found in the plant extracts was accomplished using UV-Vis (Shimadzu, Model UV-2401 PC) analysis.Te compounds with σ-bonds, π-bonds and lone pairs of electrons, chromophores, and aromatic rings were identifed using the UV-visible spectra.Table 2 shows the observed absorption bands for each plant extract.
In the UV-Vis spectra (Figure 2), the appearance of one or more peaks in the region from 200 to 400 nm is a clear indication of the presence of unsaturated groups and heteroatoms such as S, N, and O [42].Te UV-visible spectra of BO/seed/ethanol extract showed a peak at 242.1 nm that indicated the existence of favonoids [43].Peaks at 235.4 nm, 327.5 nm, 401.5 nm, and 665.4 nm in BO/stem/ethanol extract indicated the presence of steroid, terpenoid, and extended conjugation due to the presence of favonoid.

ATR-FTIR Analysis.
Te functional group of the active components was found by looking at the peak value in the infrared part of the FTIR spectrum (Perkin Elmer, Spectrum-2).In Table 3, FTIR peaks for two diferent parts of BO extracts are reported.
Figure 3 shows that the BO plant extracts of two diferent parts such as stem and seed revealed peaks at 881 cm −1 that correspond to the -CH 2 -rocking group which showed the existence of carbohydrate.Both parts of this plant extract showed peaks at 1023 cm −1 which correspond to the -C-Ogroup that revealed the presence of favonoids and cardiac glycosides.BO/seed/ethanol plant extract presented peaks at 1449 cm −1 and 1654 cm −1 which represented the -CH 3 group and -C�C-group, peak at 2943 cm −1 was responsible for -C-H-stretching of alkane that indicated the presence of terpenoids.In BO/stem/ethanol extract, there was no peak at 1654 nm which indicated that there is no presence of terpenoids which was further proved in the preliminary phytochemical screening section.Peaks at 2832 cm −1 were found in both parts of this plant extract, indicating -N-H-stretching that directed the presence of alkaloids.BO plant extracts of two diferent parts also displayed peaks at 3319 cm −1 , 3325 cm −1 that correspond to the -OH-group which indicated the presence of phenolics and favonoids [44].

Antimicrobial Activity.
Antimicrobial drugs are substances, either naturally occurring or synthesized, that prevent or eradicate the growth of microorganisms such as bacteria, fungi, helminths, protozoa, and viruses.Antimicrobial drugs can be categorized based on how they combat microorganisms.Tese include compounds that depolarize cell membranes, prevent bacteria from forming cell walls, halt protein synthesis, inhibit nucleic acid synthesis, and disrupt metabolic processes.Some basic phytoconstituents are responsible for antimicrobial activity, e.g., alkaloids, favonoids, and glycosides [45][46][47][48].
We tested the ethanolic extracts of two parts of BO for their antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi.Disc difusion method was used for antimicrobial activity testing.All the plant's extract exhibited good antimicrobial properties against the microorganisms (Table 4).
Extracts demonstrated varying levels of activity against all microorganisms.Te diameter zone of inhibition values for the extracts against all microorganisms were ≥10 mm.Te zone of inhibition values ≥8 mm for medicinal plants is considered active.Te diameter zone of inhibition varies from 10 mm to 14 mm.Based on the diameter of zones of inhibition, antimicrobial activity is categorized as inactive (<8 mm), less active (≥8 to <12 mm), moderately active (≥12 to <20 mm), and highly active (≥20 mm) [49].Te inhibition observed on the tested strains of bacteria shows that orchid extracts have a broad spectrum of activity of plant extracts against diferent strains of bacteria, which has been reported in other studies [50,51].
For conventional and traditional medicine to be able to use the active compounds in the orchids that this study focused on, they need to be better understood and tested.Also, all of the tested extracts showed diferent levels of antimicrobial properties that fght infections caused by microorganisms.

Brine Shrimp Lethality Bioassay.
Cytotoxic pharmaceuticals (also known as antineoplastics) are cancer-treating medications that introduce toxic substances to cells, blocking their reproduction or development.Tey can also treat rheumatoid arthritis and multiple sclerosis, among other conditions.Conventional cancer treatments are thought to work by killing only tumor cells or stopping their growth in a way that can't be undone.Cytotoxic medicines stop DNA from being made or damage DNA chemically, which kills tumor cells.Te nauplii of brine shrimp are simple animals that can be used to measure how deadly something is [34].It is a safe and cheap way to fnd out if a chemical or plant product is harmful to cells [52].It is also the primary tool for detecting antitumor properties [34].Teir LC 50 values can evaluate the toxicity of plant extracts.If LC 50 values are lower than 1000 μg/mL, it is cytotoxic [34].In contrast, LC 50 values of stem and seed BO showed 39.199 µg/mL and 10.659 µg/mL chronologically and standard vincristine sulfate showed 0.84 µg/mL (Table 5).Despite having lower activity than vincristine sulfate, those two components of BO extract can be considered potent cytotoxic drug holders.
Some studies confrmed that cytotoxic activity is responsible for secondary metabolites such as phenolics, favonoids, and terpenoids [53][54][55][56][57][58][59].Te presence of terpenoids Evidence-Based Complementary and Alternative Medicine      Evidence-Based Complementary and Alternative Medicine in the stem extract may explain why the stem of BO is more potent than the seed.Te stem and seed of BO also contain favonoids and phenols, which may also be responsible for exerting this cytotoxic efect.

Trombolytic Activity In Vitro.
Trombolysis, also called thrombolytic therapy, is used to break up large blood clots, get blood fowing again, and protect organs and tissues from damage.Trombolytic drugs break up blood clots by turning on plasminogen, which causes plasmin to split into two parts.Plasmin is a proteolytic enzyme that may break crosslinks between fbrin molecules, which are responsible for the structural stability of blood clots.Te results of the percentage of clot lysis of the ethanolic extracts of diferent parts of BO (stem and seed), the positive control (streptokinase), and the negative control (water) are shown in Figure 1.For positive control (100 μL of streptokinase 30, 000 IU), the clot lysis was 76.15 ± 1.94% and 92.59 ± 2.35% after 1.5 h and 24 h incubation.In contrast, in the case of negative control (water), the clot lysis was found at 3.59 ± 0.46% and 9.14 ± 1.05% after 1.5 h and 24 h of incubation, respectively.
It is seen from Figure 4 that 1000 ppm and 100 ppm solution of the seed of BO showed 20.27 ± 2.01 and 14.29 ± 1.03 after 1.5 hours and 36.92 ± 1.02 and 23.89 ± 1.49 after 24 hours of incubation period, respectively.Again, the concentration of the stem of BO was 1000ppm and 100ppm, percent clot lysis was 25.88 ± 2.01 and 16.24 ± 1.03 after 1.5 h and 44.64 ± 1.02 and 27.84 ± 1.49 after 24 h incubation time respectively.
Some studies indicate that thrombolytic activity is probably due to the diverse composition of plant extracts' phytoconstituents, including alkaloids, favonoids, tannins, and terpenoids [60,61].Te stem of BO is more active compared with the seed of BO.It may be due to the presence of terpenoids in the stem extract.

3.7.
In Vitro Antiarthritic Activity.Antiarthritic medicine treats or prevents arthritic symptoms such as joint pain and stifness.Depending on the type of antiarthritis drug, it may help in pain, reduce infammation, or weaken the immune system.Denaturation of proteins may contribute to the production of auto antigens in some arthritic conditions.Changes to electrostatic, hydrogen, hydrophobic, and disulfde bonds are likely part of the denaturation pathway [62].
In vitro antiarthritic activities of ethanolic extract of seed and stem of BO were observed.Figure 5 represents the data on the antiarthritic activities of stem and seed of BO plant extracts.Te percent inhibition of protein denaturation of 500 ppm plant extract of the stem and the seed of BO showed 53.14 ± 2.87% and 44.66 ± 0.89%, respectively, and standard showed the maximum of 85.10 ± 0.45%.A 250 ppm plant extract of the stem and seed of BO inhibited protein denaturation by 42.07 ± 0.94% and 30.03 ± 1.76%, respectively, while the standard inhibited protein denaturation by 73.79 ± 0.73%.
Te percent inhibition of protein denaturation of 125 ppm plant extract of stem and seed of BO showed 33.70 ± 1.53% and 21.06 ± 2.52%, respectively, Some phytochemical constituents have the same properties, such as favonoids, which have antirheumatism, antihypertensive, antimicrobial, diuretic, and antioxidant properties.Some phytochemicals have the same efects.For example, favonoids can treat rheumatism, high blood pressure, infections, make you pee, and fght free radicals [63,64].Both the stem and the seed of BO contain favonoids, but the stem may contain a comparatively higher amount of favonoids than the seed according to its activity.
Te results of our study show that extracts of BO could be used as antimicrobials, in chemotherapy, to break up blood clots, and to stop the body from making autoantibodies.Our research shows that extracts from diferent parts of the BO have low to moderate antimicrobial, cytotoxic, thrombolytic, and antiarthritic activity.Still, these could be an important source of antibacterial, antifungal, anticancer, antitrauma, thrombolytic, and antiarthritic elements.

Conclusion
In this study, 14 microorganisms were used, and orchid samples were compared to ciprofoxacin as a reference.It was discovered that the BO/seed extract had signifcant antibacterial activity, compared to BO/stem extract.Te LC 50 values were evaluated using the brine shrimp lethality assay.Te BO/stem extract demonstrated a more destructive efect than the BO/seed extract, measuring LC 50 value for 10.659 μg/mL.To evaluate the extracts' thrombolytic activity, two concentrations (1000 and 100 ppm) and two incubation periods (24 and 1.5 hours) were used.In terms of its thrombolytic activity, the BO/stem extract had shown  8 Evidence-Based Complementary and Alternative Medicine more potential.Furthermore, the highest values for protein denaturation were found at 500, 250, 125, and 62.5 ppm (53.14 ± 2.87%, 42.07 ± 0.94%, 33.70 ± 1.53, and 19.45 ± 1.08%), indicating the antiarthritic efcacy of the BO/stem extract to other parts.Our study will be helpful to fnd novel phytochemicals in light of the emergence of drugresistant microorganisms, cancer, stroke due to blood clots, autoimmune disease, etc. Positive results obtained from diferent investigations mentioned above run on diferent organs of orchid were provided us a primary indication of its efectiveness as antimicrobial agents, antitumor, antiarthritic, and thrombolytic compounds.Further research on this plant is needed.Tis study could play a vital role in fnding a lot of sources to invent drugs for the treatment of diseases related to antimicrobial, cytotoxic, thrombolytic, and arthritic activity as candidates for the future in vivo investigation.

Figure 5 :
Figure 5: Antiarthritic activity of ethanolic extracts of BO stem and seed compared to standard (diclofenac sodium).
Gelatin Test: 0.2 g of extracts and 3 mL of 1% gelatin containing 10% NaCl (a few drops) were added to test tubes.Te presence of white precipitate suggests the presence of tannins.
2.2.6.Test for Saponins.Froth Test: 0.1 g of each extract was taken in test tubes and diluted with 15 mL of distilled water, and the mixture was shaken vigorously for 15 minutes.Te formation of a 1 cm layer suggests the presence of saponins.2.2.7.Test for Phenols.FeCl 3 Test: 0.2 g of each extract was taken in test tubes and boiled with 5 mL of 45% ethanol for 5 min.Ten, the mixture was cooled and fltered.After that, 5 mL of distilled water and three drops of 5% FeCl 3 (w/v) were added with 1 mL of the fltrate.A transient greenish to black coloration confrms the presence of phenols.2.2.8.Test of Tannins.2.2.9.Test for Carbohydrates.Molisch's Test: 0.1 g of extracts and 5 mL of distilled water were taken in each test tube.
[41]40]y.Trombolytic properties were investigated according to the method disclosed in Prasad et al.[39,40].Remi-R-83 A) operated at 2000 rpm for 1 minute.After that, the serum was entirely separated without disturbing the clot, and reweighing each falcon tube was done to measure clot weight.All the falcon tubes containing clots were labeled correctly, and 100 µl of diferent plant extracts, 100 µl of SK (Positive Control), and 100 µl of distilled water (Negative Control) were added to the falcon tubes.Ten, falcon tubes were kept in an incubator at 37 °C for 90 minutes and 24 hours to monitor the clot lysis.Te dissolved clot was carefully taken out, and after 90 minutes and 24 hours of incubation, the gravimetric method was used to fgure out the clot lysis percentage.Tis test was done in triplicate.Te method described in Pavithra et al.[41]was used to keep track of arthritic activities.
2.7.1.Procedure.Te collected blood was allowed to clot in falcon tubes for around ffteen minutes.Ten, the serum was separated from the blood using a centrifuge machine (Model:

Table 1 :
Diferent phytochemicals found in ethanolic extracts of diferent parts of BO.

Table 2 :
UV-visible peaks of diferent plant extracts.

Table 3 :
FTIR peaks of two diferent parts of BO extracts.

Table 4 :
Antimicrobial activity of ethanolic extracts of diferent parts of BO.

Table 5 :
Brine shrimp lethality bioassay of ethanolic extracts of diferent parts of BO.
Figure 4: Trombolytic properties of ethanolic extracts of stem and seed of BO compared with standard (streptokinase).