Influence of Storage Temperature and Duration on Phytochemical Contents and Antioxidant Activities of the Inflorescence of Aranda Hybrids

Utilization of natural products from plants for health, medical and beauty-related industries is trendy and increasingly in demand. Orchids are reported to be rich in antioxidant properties. They can be used for medicinal purposes such as antioxidant, antirheumatic, anti-inflammatory, anti-carcinogenic, hypoglycemic, antimicrobial, anticonvulsive, anti-tyrosinase activity, relaxation, neuroprotective, antivirus, etc. (Gutiérrez, 2010, Rungsang et al., 2023).
               
Aranda hybrids (formerly called ‘Mokara’) are monopodial orchids. They are progenies from crossing three orchid genera, including Arachnis, Ascocentrum and Vanda. They have long, parallel-veined, leathery leaves arranged on two opposite sides along the stem from the base to the top of the plant. As a famous cut flower, the inflorescence of Aranda hybrids is about 30-50 cm long, composed of 9-19 flowers. Each flower is about 5-8 cm wide and has a long vase life of about 5.8-22 days, depending on the cultivar (Dymek et al., 2019). Aerial roots are produced from the axillary buds of internodes. As a climbing orchid, the plant can grow upwards and reach a height of 1.5-2.0 meters when fully mature. The matured plant has a long stem with numerous leaves and aerial roots. Growers need to top-cut the mature Aranda’s stem to prevent it from growing too tall, which can make it difficult to harvest the inflorescence. The removed leaves and aerial roots can be used for extracting bio-substances for medicinal purposes without harming the mother plants.  Some biological screenings exist on stems, roots and leaves of monopodial orchids such as Vanda coerulea and some Phalaenopsis hybrids. The crude hydro-alcoholic stem extract of V. coerulea showed high DPPH/•OH radical scavenging activities that can reduce skin oxidative stress from UV exposure. The extract can inhibit cyclo-oxygenase 2 (COX-2) activity, an enzyme for generating prostaglandin metabolites and cause skin inflammation (Simmler et al., 2010). In Phalaenopsis hybrids, the ethanol extracted from roots displays higher DPPH/•OH radical scavenging activities than the leaf part. However, the highest total phenolics and total flavonoids were detected from the leaf part. The roots of Phalaenopsis can be harvested from the decorative finishing pots to reduce waste. They serve as a potential source of natural antioxidants (Minh et al., 2016). Although the Aranda is a well-known commercial orchid, the information about the antioxidant properties of these orchids is limited. Accordingly, this study reports the Aranda hybrids’ total flavonoid, phenolic contents and antioxidant activities. Furthermore, the influence of storage temperature on the antioxidant properties of the dried samples of the Aranda hybrids under long storage for six months was investigated. We hypothesize that the root part of the Aranda hybrids likely contains the highest level of bioactive substances and antioxidative activities, as reported in some monopodial orchids like the Phalanopsis (Minh et al., 2016). Additionally, storing the samples in cool conditions for six months may help maintain better antioxidant properties compared to storing them at the room temperature. The finding will provide valuable information for the medicinal utilization of these orchids to add value to the raw materials.

Plant materials and extraction procedure
 
Five-year-old Aranda hybrids were used in this study consisting of Aranda Bangkhuntian Gold, A. Calypso, A. Royal Sapphire and A. Sayan Duangporn (Fig 1). The orchids were grown at a commercial farm in Kratumban district, Samut Sakhon province, Thailand. The orchid parts, including the leaves, roots, floral buds and blooming flowers, were collected, cleaned with distilled water and dried at 70°C for 48 hours in a hot air oven. Dried sample powders were separated to keep at room temperature (25-28°C) and cool temperature (5-7°C) by following experimental treatments. For the preparation of the extracts, one gram of dried sample powder was macerated with 10 mL of ethanol (80%) and incubated at room temperature (25°C). After 24 h, the extracts were filtrated with Whatman® paper filter no. 1.  The extracts were diluted with 80% ethanol to be a final concentration of 1000 µg/mL for determining phytochemical contents (total flavonoids and total phenolics) and antioxidant properties (ABTS, DPPH and Fe (II) scavenging activities).  
 


Determination of total flavonoid contents
 
Total flavonoid content was determined according to the colorimetric assay modified by Djeridane et al., (2006). The 100 µL of Aranda extracts at a concentration of 1000 µg/mL was mixed with 100 µL of aluminum chloride (2% w/v) in a 96-well plate and incubated at ambient temperature for 15 min. Then, the solution was measured at an absorbance of 430 nm using a microplate reader (Multiskan Go, Thermo Scientific, MA, USA). The total flavonoid content in each extract was compared with the rutin standard curve when Y= 0.007x - 0.003 (R2= 0.999). The result was expressed as mg of rutin equivalents (RUE) per g of extract (mg RUE/g extract).
 
Determination of total phenolic contents
 
The modification method from Siddhuraju and Becker (2003) determined the total phenolic content. The 20 μL of Aranda extracts were mixed with 50 μL of distilled water and 50 μL of Folins-phenol reagent (ratio of reagent: water 1:1) in a 96-well plate, then added with 80 μL of sodium carbonate (7.5% w/v). The solution was incubated at ambient temperature for 30 min and measured at an absorbance of 725 nm using a microplate reader. Gallic acid (0.001 to 0.01 mg/mL) was used as a standard. The total phenolic content in each extract was compared with the standard curve when Y=0.0079x + 0.055 (R2=0.997). The result was expressed as mg of gallic acid equivalents (GAE) per 1 mg of solid crude extract (mg GAE/g extract).
 
Determination ABTS scavenging activity
 
The total antioxidant activity of the ethanol extracts was determined by ABTS assay (Hsu et al., 2011) with a slight modification. A mixture of 2,2¢-azinobis (3-ethylbenzothiazoline -6-sulfonic acid (ABTS) and potassium persulfate were incubated in the dark at room temperature (25°C±2) for induced oxidation to be ABTS·+ for 16 hr. It measured the mixture at an absorbance of 734 nm until the absorbance value reached 0.700±0.050. Added the ethanolic Aranda extract to the mixture, kept in the dark for 6 min and measured at an absorbance of 734 nm. The tert-butylhydroxytoluene (BHT) (Sigma-Aldrich, Inc., USA) and tocopherol were used as positive control for comparing antioxidant capacity.
     
Determination DPPH scavenging activity
 
The total antioxidant activity of the ethanol extracts was determined by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with a slight modification (Boskou et al., 2006). A hundred micro milliliters of Aranda extract were mixed with 40 μL of DPPH solution (Sigma-Aldrich, Inc., USA) at 0.5 mM and 100 μL of sodium acetate buffer (0.1 M, pH 5.5). Then, the solution was kept in the dark at room temperature (25°C±2) for 30 min and measured at an absorbance of 517 nm. The tert-butylhydroxytoluene (BHT) (Sigma-Aldrich, Inc., USA) was used as a positive control for comparing antioxidant capacity.
  Where:
O.D.= Optical density in the presence or absence of the samples.
 
Determination Fe (II) scavenging activity
 
The reducing ability of the ethanolic extracts was determined following the methods of Oyaizu (1986) with slight modification. The 400 μL ethanolic Aranda extract was added to 800 μL of 1% potassium ferricyanide. Then, the solution was incubated at 50°C for 20 min, added with 800 μL of 1% trichloroacetic (TCA) and centrifuged at 5,000 rpm for 10 min. It took only 400 μL of supernatant to mix with 400 μL distilled water and 80 μL of 1% ferric chloride. The solution was measured at an absorbance of 700 nm using the tocopherol as a positive control for comparing antioxidant capacity.
 
Statistical analysis
 
Statistical analysis was performed using one-way ANOVA. The data were presented as mean±standard deviation.  Mean comparisons were analyzed using Duncan’s multiple range test (DMRT) at p<0.05.  All analyses were performed in four biological replicates.

Phytochemical contents and antioxidant activities of Aranda after harvested
 
After harvesting and separating Aranda’s leaves, roots, flower buds and blooming flowers, the samples were dried and promptly analyzed for biological substances and antioxidant activities. The results showed that the total flavonoid content was markedly detected in the leaf part of all Aranda hybrids, ranging from 26.63-40.05 mg RUE/g extract, which was 1.48-1.96 folds higher than the roots and the other floral parts. Similarly, the total phenolic content was greatly accumulated in the leaves part, ranging from 152.00-254.13 mg GAE/g extract, 1.23-2.50 folds higher than the floral parts (Table 1). The highest total flavonoid content in leaves has also been reported in aqueous extract of Thapsia garganica L. (Djahida and Houcine, 2021) and the ethanolic extract of Dendrobium sulcatum, in which both the stem and leaf extracts displayed a higher level of total flavonoids than the floral organs (Rungsan et al., 2023).  In response to environmental stress, some mature orchids accumulate higher levels of total phenolic contents than young ones (Obsuwan et al., 2019). In the same way, the mature Aranda used in this study showed a high level of total phenolic content.
       


Interestingly, in the A. Bangkhuntian Gold, the total phenolic content was detected in a major proportion in the root part as 291.67 mg GAE/g extract, which is remarkably higher than other organs of another three cultivars, which have only 97.44-254.13 mg GAE/g extract (Table 1). Compared among the four plant parts, the floral bud and the blooming flowers have moderated equally levels of the total flavonoid and total phenolic contents ranging from 8.00-20.80 mg RUE/g extract and 97.44-143.44 mg GAE/g extract, respectively (Table 1). 
       
In most of the Aranda hybrids, the highest ABTS and Fe (II) antioxidant activities were detected in the leaf part, with values ranging from 53.13 to 78.85% and 11.19 to 15.07%, respectively. These values were 1.13 to 2.78 times higher than the antioxidant activities observed in the floral parts (Table 1). The DPPH activity of most Aranda hybrids was highest in the root part (67.37-80.90 %), which was 1.27-1.58 times higher than in the floral parts (Table 1). The ABTS and DPPH radical scavenging showed a positive correlation (r=0.762). Low levels of the Fe (II) antioxidant activity in this research indicated that the ethanolic extract of Aranda samples may not be favorable for this assay.  Interestingly, as the total phenolic compounds are plant secondary metabolites for antioxidation, there was a strong correlation between the total phenolic contents and the ABTS antioxidant activity (r=0.90). Minh et al., (2016) reported that eleven phenolic compounds had been identified from Phalaenopsis hybrids such as protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, vanillin, ferulic acid, sinapic acid, p-coumaric acid, benzoic acid and ellagic acid. The strong correlation between the total phenolic content and total flavonoid content with the total antioxidant capacity was also reported in naked barley (Bechiri et al., 2024) and Ajuga iva (Lahrizi et al., 2024).
       
Although the four Aranda hybrids have diverse petal colors (pink, deep pink, yellow and deep yellow), there are minor differences in the total flavonoid, total phenolic contents and antioxidant activities among the variations in flowering shades. Therefore, the flowering stages (bud and blooming) or petal shade do not influence the total flavonoid and total phenolic contents and antioxidant activities of the Aranda hybrids. Among the Aranda hybrids, A. Bangkhuntian Gold exhibited the highest inhibitory effect on ABTS and DPPH radical scavenging in the root part (80.90-82.23%) (Fig 4-5), which correlates with the highest level of total phenolic contents in the roots. The richness of the total phenolic and antioxidant activities in the root of A. Bangkhuntian may be attributed to species specificity. Aranda is a monopodial orchid that grows upward, with a stem that is difficult to harvest for its floral inflorescences. Therefore, the orchid growers usually prune by cutting the top shoot of the aging Aranda to maintain a suitable level of plant height. Therefore, growers can benefit from the cut-off shoot, which consists of numerous leaves and aerial roots that can be used as a material for bio-substance extractions.
   
Phytochemical contents and antioxidant activities of Aranda after storage for six months
 
The dried powder of Aranda was kept at room temperature (25-28°C) or in a refrigerator (5-7°C) for six months to investigate the influence of long-term storage and temperature on the phytochemical contents and antioxidant capacities. The results showed that the total flavonoid content in the leaves and roots of all Aranda hybrids was sensitive to storage at room temperature, as the total flavonoid level significantly decreased under room temperature conditions (34.80-155.58 mg RUE/g extract) compared to cool storage (71.83-241.49 mg RUE/g extract) (Fig 2). The study involved storing floral buds and blooming flower parts as storage samples for six months at either room or cool temperatures. The total flavonoid content in these samples ranged from 69.25-118.25 and 71.83-126.79 mg RUE/g extract, respectively. The results showed no significant difference in the flavonoid content at P<0.05 between the two storage conditions (Fig 1). Flavonoids are reported to be heat-sensitive substances. The rapid water loss during processing or prolonged exposure to sunlight of the samples can result in a decrease in the flavonoid content. In Dryopteris erythrosora leaf pretreatment by sunlight has a faster rate of water loss in dried samples, resulting in a marked reduction of total flavonoid level compared to shade drying (Zhang et al., 2019). In this study, the floral buds or blooming flowers have lower water content and the rate of water loss may be slower than in the leaves and the roots. Therefore, the flavonoid contents can be better maintained in long-term storage. The total phenolic contents from different parts of four Aranda hybrids were found to be at similar levels when stored under both conditions (Fig 3). It means that the room temperature has less influence on the total phenolic contents of the orchids. Similarly, the storage at room temperature or in a refrigerator had a non-significant influence on the ABTS, DPPH and Fe (II) radical scavenging of all parts of most Aranda hybrids (Fig 4-6), except for the A. Royal Sapphire stored in cool storage can maintain better ABTS activity in flower buds and blooming flowers (Fig 3).    
       








The overall results suggested that the 25-28°C storage for six months has little affected the total phenolic contents and antioxidant activities compared to the cool storage because the dried samples lost their antioxidant properties slowly. Therefore, the dried sample can be kept at room temperature if the cool temperature facility is unavailable for at least six months.

The four Aranda hybrids include A. Bangkhuntian Gold, A. Calypso, A. Royal Sapphire and A. Sayan Duangporn have the highest total flavonoids and phenolic contents, the ABTS and Fe (II) antioxidant activities in the leaves part, which are remarkably higher than the floral parts. However, the DPPH activity of most Aranda hybrids was the highest accumulated in the root part. Compared among the four Aranda hybrids, the A. Bangkhuntian Gold has the highest total phenolic contents in the leaf and ABTS and DPPH antioxidant activities in the root. The six months of dried samples storage at room temperature can maintain the total phenolic contents, the ABTS, the DPPH and Fe (II) antioxidant activities compared to the cool storage. However, the total flavonoid contents were decreased when stored under room temperature conditions.

We highly appreciate the Oranee garden ang orchid farms supplying the orchid materials used in this study.

The authors declare that there are no conflicts of interest.