DOI: 10.31038/IMROJ.2021625

Introduction

Serious and continuing illnesses have risen to prominence everywhere with generally increased life expectancy and somewhat fewer threats to health and life due to acute illness [1]. Health care systems that invest heavily in acute hospitals are recognising the need to repurpose services to include care beyond their walls to where people with prevalent chronic illnesses live. Multiple services – both medical and social and often low-tech – are required by patients with multiple morbidities, not the stock-in-trade of many major hospitals.

Now, the intention is to provide care for the patient as an individual rather than as a recipient of separate programs of care for his or her several separate medical problems (arthritis, heart disease and mild dementia). Anecdotes tell of four cars parked outside the home of a patient with multimorbidity, each bearing a therapist for one aspect of the patient’s problems. Chronic diseases such as those mentioned are the leading cause of death in Australia, with over 51% of hospitalisations and 89% of deaths associated with chronic disease [1]. They also cause a large out-of-pocket burden for many, with as many as 78% of Chronic Obstructive Pulmonary Disease (COPD) patients experiencing economic hardship while managing their illness [2]. Many chronic diseases are preventable [3], but despite this, preventative health makes up only 1.3% of all health spending [4], begging the question ‘Is it time we evaluated our current system?’.

‘Integrated care’ refers to the concern to improve the overall patient experience, from prevention to post-discharge care, and attain both greater value and efficiency from our health delivery model. It aims to address the fragmentation which often occurs in patient services; through integrated care the hope is that now the patient will benefit from a more holistic, joined-up service so that useless duplication and inappropriate admissions to hospital will be reduced.

In Australia, which enjoys high levels of health and generous financial investment in health care, much experimentation has occurred with different configurations of integrated care (as this joined-up pattern of hospital and community care is termed). In western Sydney, two programs (among many) conducted in recent years give insights into what can be done with modest investment and decisive management to improve the quality of life of patients with multiple chronic problems. The two small examples with which we are familiar, do not provide a clear set of directions about what to do in providing integrated care. Rather, because of our marginal involvement in them we been able to observe their operation and note what works well.

The Respiratory Ambulatory Care Service (RACS)

To assist patients with severe chronic obstructive lung disease (and usually other problems as well), this program was established by Professor John Wheatley, a respiratory physician at Westmead Hospital, ably assisted by Mary Roberts, a nurse practitioner, at Blacktown Hospital in 2001 with NSW Health Chronic and Complex Care funding. Over the years it was modified to meet the needs of the population [5]. The program had 8 pulmonary rehabilitation classes a day across Western Sydney Local Health District, with 12 patients enrolled in each class, with a text message support service which had over 100 patients enrolled in the Breathlessness Clinic. These patients were assessed at Blacktown Hospital by a physician, a physiotherapist, and a nurse.

Initially starting as a pure pulmonary rehabilitation program, it developed into a comprehensive COPD management program with a 24-hour helpline with COPD action plans [6]. A ten-week program of exercises and support was arranged for each patient. They were then cared for at home by the ambulatory team with regular visits by the nurses. A laptop-based medical record was maintained for each patient. The laptop passed to the duty nurse each night. Each patient was encouraged to contact the team by phone if they had a problem, and many did, especially at night when an attack of breathlessness might lead to panic which, without the support of the RACS, usually led to an ambulance call to take them to the hospital emergency department.

A conversation at 2 am with a nurse who knew the patient and had their electronic record in front of them on their laptop, could help decide if hospital admission (the default option in the absence of someone to speak to, with massive social dislocation to the patient and expense to the health service) is required or whether the patient can be ‘talked down’ and reassured to go back to bed, their problem to be fully explored, at home, in daylight. Past patients also had access to this 24-hour helpline which was utilised by patients who had not been in years, and a monthly subscription newsletter for education and information.

The program has been fully described in publications [6] SRL assisted for two years as a respiratory physician, servicing a clinic once a week and occasionally accompanying the nurses on home visits.

The program was established by Wheatley because he saw how patients once beyond the hospital could easily flounder for lack of specialised support. General practitioners often did not have the time or the access to resources available to the RACS team. By maintaining strong links between the specialists and the nurse led RACS team, integration was achieved. The results of the program were impressive with hospital admissions halved among program participants in the year after enrolment compared with the year before. Furthermore, during ongoing the COVID-19 pandemic, RACS was altered to provide telehealth and tele-education, as well as text messaging support. This not only is a great example of how such a system can evolve to suit the needs of the population, but a potential glimpse into the future.

Perhaps the most important caution is that health service managers should not be seduced into supporting integrated care programs because they believe they save money (they may) but rather should support them because they lead to a better quality of life for enrolled patients, albeit often at increased cost. It is misleading in the long-term to promise without evidence that ‘integrated care reduces hospital costs by keeping people out of hospital.’

Worldwide, the demand for hospital beds is such that if a bed is emptied because a chronically ill patient is cared for in the community instead, it is quickly filled by someone else (say, with a femoral neck fracture) and the costs to the health system remain unchanged. Clinicians know this and their interest in integrated care is because it is good medicine, not because it is cheap. In recruiting clinical staff to integrated care programs it is important to make this point strongly.

The Western Sydney Integrated Care Demonstrator Project

In 2016, the New South Wales (NSW) Ministry of Health provided affirmative funding to several health districts to support demonstration projects in integrated care. An allocation to the Western Sydney Local Health District enabled the development, again with a nurse manager and this time an extensive team of health professionals of different disciplinary skills and interests from both the hospital and the community, in relation to the care of patients with diabetes, heart disease and cardiovascular problems.

Although these three disease themes were developed relatively independently, they were linked through patient recruitment and documentation. Hospital specialists were critical to the development and implementation of the project as were local general practitioners. Nurses played a pivotal role in making the program happen, assisting greatly in the recruitment of patients both in Westmead Hospital and the western Sydney community through general practice.

This was a more complex program than RACS and required more managerial effort because of the number and diversity of practitioners involved and because the motivation for the program was ‘top down’, i.e., it came from NSW Health primarily rather than from practitioner concerns for better care of their patients. Practitioners needed education and support to be persuaded to participate.

The program used several strategies, including care facilitators, extensive use of IT, action plans for specialist care, shared care plans, the establishment of a rapid access and stabilisation service for patients that avoided them having to use the Emergency Department if they deteriorated, support payments to general practices to enrol and document the progress of enrolees, and promotion of the concept of a patient centred medical home.

Qualitative evaluation of the program was conducted, and the results are detailed in two papers by the principal evaluation research person [7,8]. SRL chaired the evaluation research advisory committee for the program.

Conclusion

There are several takeaway messages from these two programs. First, there is a big challenge in bringing the multiple players, both in the community and in the hospital, into a harmonious and productive team. This requires sensitivity and professional management. Effective integrated care relies on negotiation among several professional groups and separate government agencies, e.g., health and social services. Second, the recruitment of patients to these programs necessitates patient explanation and a responsiveness to questions that patients will have about their continuing care. People who are sick are frequently anxious not only about their health but also about the adequacy and dependability of their care. Familiarity with one or more carers, often a nurse, is important to achieve continued participation by patients and their carers.

Third, integrated care programs must be well organised and managed and this requires resources both in terms of workforce and money. The lines of responsibility (and accountability) are often long and intertwined and it is unusual for this arrangement to proceed smoothly in every respect. Negotiation is needed at many points in the program and once again time and patience are needed. The development of tailored IT arrangements to enable integrated care is critical and time-consuming.

Fourth – and there is a serious risk here – the program may be hijacked by managers who are ignorant of the detail of effective integrated care and see it as a way to save money. This is implausible [9] and can lead to program failure. Advocacy for the program is essential at all stages. In this regard it is very helpful to have leadership in the management of the program by senior and respected clinicians, both in the hospital and also in the community.

Fifth, the evaluation of integrated care programs is difficult because of the complexity of the relations among different groups of players. Comparison of patients managed conventionally and via integrated care is hard to engineer to the rigorous standards of a randomised controlled trial. The outcomes desired from integrated care often have to do with marginal improvement in quality of life. Even using an end-point such as reductions in hospital admissions can miss the point: some patients may need more rather than fewer admissions if the integrated care program brings to light problems that were previously not managed or not managed well.

In conclusion, the use of integrated care for the optimal management of patients with complex, serious, and continuing illness offers advantages especially for those living outside institutions and not in hospitals or care homes. An effective integrated care program should include aspects of an increased focus on preventative and population health, changes to the patients experience while admitted in hospitals, and higher levels of community support and care for patients outside of hospital walls [10]. Although in its early stages of development, it is attracting great interest among patients, their carers and families, and clinicians seeking to offer them the best quality of care.

References

1. Australian Institute of Health and Welfare 2020. Australia’s health 2020: in brief. Australia’s health series no. 17 Cat. no. AUS 232. Canberra: AIHW.
2. Essue B, Kelly P, Roberts M, Leeder S, Jan S (2011) We can’t afford my chronic illness! The out-of-pocket burden associated with managing chronic obstructive pulmonary disease in western Sydney, Australia. J Health Serv Res Policy 16: 226-231. [crossref]
3. Australian Institute of Health and Welfare 2014 Australia’s health 2014. Australia’s health series no. 14. Cat. no. AUS 178. Canberra: AIHW.
4. HJ, AS (2017) Preventive health: How much does Australia spend and is it enough? Canberra: Foundation for Alcohol Research and Education.
5. Smith TA, Roberts MM, Cho J-G, Klimkeit E, Luckett T, et al. (2020) Protocol for a Single-Blind, Randomized, Parallel-Group Study of a Nonpharmacological Integrated Care Intervention to Reduce the Impact of Breathlessness in Patients with Chronic Obstructive Pulmonary Disease. Palliative Medicine Reports 1: 296-306.
6. Roberts MM, Leeder SR, Robinson TD (2008) Nurse-led 24-h hotline for patients with chronic obstructive pulmonary disease reduces hospital use and is safe. Intern Med J 38: 334-340. [crossref]
7. Trankle SA, Usherwood T, Abbott P, Roberts M, Crampton M, et al. (2019) Integrating health care in Australia: a qualitative evaluation. BMC Health Services Research 19: 954.
8. Trankle SA, Usherwood T, Abbott P, Roberts M, Crampton M, et al. (2020) Key stakeholder experiences of an integrated healthcare pilot in Australia: a thematic analysis. BMC Health Services Research 20: 925. [crossref]
9. Nolte E, Pitchforth E (2014) What is the evidence on the economic impacts of integrated care? Copenhagen Ø, Denmark: World Health Organisation.
10. Baxter S, Johnson M, Chambers D, Sutton A, Goyder E, et al. (2018) The effects of integrated care: a systematic review of UK and international evidence. BMC Health Services Research 18: 350. [crossref]

DOI: 10.31038/IMROJ.2021624

Abstract

In performing molecular profiling of secondary metabolites, a lot of research has focused on biogenic volatile organic compounds with medium to low polarity. In this study, chemical composition similarity relationships among the various organs of the Ilex cornuta Lindl. & Paxton were assessed based on the analysis of hydrophilic volatile compounds. GC-MS analysis was conducted to characterize and classify the chemical compounds. A total of 36, 46, 42, 25, 64, 26 compounds have been respectively extracted from the root, stem, stem skin, leaf, flower and fruit. The six organs have 3 common compounds and large percentages of exclusive compounds ranging from 36.0% to 62.5% with a mean of 49.8%, indicating substantial component differences among the different organs. The percentage of overlapping compounds between each of the two organs ranges from 10.9% to 44.0%, which is relatively small, further demonstrating the strong organ specificity of the chemical composition. The overlapping index is used to reveal the similarity among the organs. The stem shares the maximum similarity while the fruit the minimum similarity with the other organs. Aside from fruit, the average overlapping indices between each of the other two organs correlate well to their physical proximity. In conclusion, hydrophilic volatile metabolites are a class of natural products that are rarely investigated but constitute a significant part of the plant chemical composition. Chemical profiling of these metabolites could provide a valuable tool for the plant taxonomy and help understand the chemically mediated biological phenomena.

Keywords

Chemical composition similarity, GC-MS, Hydrophilic volatile compounds, Ilex cornuta Lindl. & Paxton, Plant taxonomy

Introduction

Plant taxonomy is traditionally conducted based on macroscopic and microscopic morphological characteristics. Growing evidence suggests that many biologically relevant entities could be missed in the studies that rely solely on morphological traits, particularly since speciation is not always accompanied by morphological change [1,2]. In recent years, plant chemical taxonomy has been developed to perform classification based on a wide array of biologically active secondary metabolites [3]. The expression of secondary metabolites could vary due to convergent evolution or differential gene expression [4], suggesting that the metabolite content of plants may reveal more information on the bioactive pattern of plants in comparison to morphology characterization [5].

In performing molecular profiling of secondary metabolites, a lot of research has focused on biogenic volatile organic compounds with medium to low polarity [6-9]. Volatile compounds are secreted and part of them are volatilized immediately after secretion [10,11]. The remaining part is stored in the special structure of the plant as in the case of essential oils [12-14]. Additionally, Berlinck and collaborators found that the vast majority of new compounds from natural sources reported in recent literature are compounds of medium to low polarity. Water-soluble, volatile, minor and photosensitive natural products are yet poorly known. One of the possible reasons for this trend could be that organic solvents of medium to low polarity used in isolation procedures require less time and less sophisticated instrumentation to be evaporated [15]. The author speculates that there is a class of hydrophilic volatile compounds in plants that are dispersed or dissolved in the water phase, evaporated with water vapor, and whose polarity and volatility are somewhere between essential oils and water-soluble compounds. To protect this type of ingredients from loss during extraction, water vapor distillation is used to collect volatile compounds that are dispersed or dissolved in the plant’s water phase. The root, stem, stem skin, leaf, flower and fruit of the Ilex cornuta Lindl. & Paxton were analyzed as study samples. Volatile essential oils were removed by using Soxhlet extraction method. Hydrophilic volatile compounds obtained by water reflux extraction are characterized and classified by quantitative GC-MS. The study revealed the potential use of hydrophilic volatile metabolites in the plant taxonomy and understanding the chemically mediated biological phenomena.

Materials and Methods

Material

Ilex cornuta Lindl. & Paxton was collected in Nanjing, China. Its roots, stems, stem skins, leaves, flowers and fruits were washed, cut into pieces, dried at 30°C and stored at 2-8°C prior to use.

Chemicals and Reagents

Ethyl acetate was purchased from Xilong Chemical Co., Ltd (Shantou, China). Hexane was purchased from Shanghai Titan Scientific Co., Ltd (Shanghai, China). Activated carbon was purchased from Shanghai Chemical Reagent Procurement Center (Shanghai, China). C7-C40 saturated alkanes standard was purchased from Anpel Laboratory Technologies Inc. (Shanghai, China).

Sample Preparation

Each sample was sliced and dried at 30°C. After ground into powder, the samples were sieved through 80 mesh followed by 180 mesh. Approximately 6 g of the sample were subjected to Soxhlet extractor method with hexane for 24 hrs to remove essential oils and other lipophilic compounds. The remainder was then removed and dried at 30°C in the ventilation cabinet. Approximately 4 g of the dried powder was then added into a 6 x 7 cm nonwoven bag together with three glass balls of 4 cm diameter. At least 3 segments of thread were used to separate and tighten the bag into 3 parts, each containing a glass ball and even amount of the dried powder. The bag was then placed in a flask and 2100 mL of water was subsequently added to soak the powder for about 2 hrs. After reflux extraction for 6 hrs, 2 L of distilled water was collected. The same reflux extraction was repeated to collect another 1 L of distilled water for a total of 3 L. After cooling, activated carbon (4 g) was added to absorb the active ingredients from the 3 L of distilled water for about 8 hrs. The activated carbon containing the active ingredients was then filtered and dried at 30°C for 12 hrs. Ethyl acetate was subsequently added to isolate the active ingredients from the activated carbon using Soxhlet extractor method for 8 hrs. The resulting ethyl acetate extract was left in the ventilation cabinet to dry at 30°C. The dried active ingredients were finally re-dissolved using ethyl acetate, filtrated through 0.22 µm filter and analyzed using GC-MS.

GC-MS Analysis

Analysis of hydrophilic volatile compounds was performed using Shimadzu GCMS-QP2010 Single Quadrupole GC-MS (Kyoto, Japan). A Rxi-1 ms GC capillary column (30 cm length, 0.25 mm inner diameter and 0.25 µm thick film) from Shimadzu (Kyoto, Japan) was used for analysis.

One microliter of sample was injected in split mode with split ratio of 5 to 1. GC inlet temperature is set at 280°C. High purity nitrogen (≥99.999%) was used as carrier gas in constant flow mode at 1 mL/min. The initial temperature of the GC oven is set at 60°C and held for 1 min, then ramped at 4°C/min to 160°C and held for 3 mins, followed by 2°C/min to 280°C and held for 6 mins. Finally, the temperature is raised to 300°C at 4°C/min and held for 6 mins. The mass spectrometer was operated in positive electron ionization mode at 70 eV and all spectra were recorded in full scan with a mass range of 40-700 Da. The interface temperature is set at 280°C and ion source temperature is set at 250°C.

Data Processing and Compound Identification

The GC-MS data processing was done with Shimazdzu GCMS Solution software. Compound identification was performed by applying several assignments, e.g., reference standard analysis, retention index calculation, and by NIST08 Spectrum Library comparison. Only peaks with area greater than 3 million are analyzed. The overlapping percentage is calculated by the number of overlapping compounds divided by the total number of hydrophilic volatile compounds from each of the two organ and times 100. Overlapping index is calculated by the number of overlapping compounds squared and divided by the total number of hydrophilic volatile compounds from each of the two organs. In addition, hierarchical clustering analysis was performed with Python to assess the similarities between each of the two organs by analyzing the number of overlapping hydrophilic volatile compounds.

Results and Discussion

The root, stem, stem skin, leaf, flower and fruit of the Ilex cornuta Lindl. & Paxton contain compounds that are water soluble and can volatilize with water vapor. These hydrophilic compounds do not separate from the water phase and possess greater polarity than essential oils. The largest number (64) of hydrophilic volatile compounds are isolated from the flower and the smallest (25) from the leaf, indicating that the number of hydrophilic volatile compounds varies greatly from organ to organ. The hydrophilic volatile compounds include aromatics, fatty acids, furans, heterocycle, esters, alkanes, ketones, halogens and other types of small molecular compounds. This is a diverse group of molecules that could contribute to the expression of biological information about the plant. Tables 1-6 present the lists of hydrophilic volatile compounds identified from the root, stem, stem skin, leaf, flower and fruit, respectively. The bold and italic fonts in the table are used to refer to exclusive compounds that are only found in the specific organ and not contained in any other organ.

Table 1: List of the hydrophilic volatile compounds identified from the root of the Ilex cornuta Lindl. & Paxton.

Note: The bold and italic fonts are used to refer to exclusive compounds. RT: Retention time. RI: Reflex index.

Table 2: List of the hydrophilic volatile compounds identified from the stem of the Ilex cornuta Lindl. & Paxton.

Note: The bold and italic fonts are used to refer to exclusive compounds. RT: Retention time. RI: Reflex index.

Table 3: List of the hydrophilic volatile component identified from the stem skin of the Ilex cornuta Lindl. & Paxton.

Note: The bold and italic fonts are used to refer to exclusive compounds. RT: Retention time. RI: Reflex index.

Table 4: List of the hydrophilic volatile component of the leaf of the Ilex cornuta Lindl. & Paxton.

Note: The bold and italic fonts are used to refer to exclusive compounds. RT: Retention time. RI: Reflex index.

Table 5: List of the hydrophilic volatile component identified from the flower of the Ilex cornuta Lindl. & Paxton.

Note: The bold and italic fonts are used to refer to exclusive compounds. RT: Retention time. RI: Reflex index.

Table 6: List of the hydrophilic volatile component identified from the fruit of the Ilex cornuta Lindl. & Paxton.

Note: The bold and italic fonts are used to refer to exclusive compounds. RT: Retention time. RI: Reflex index.

As shown in Table 7, the total number of hydrophilic volatile compounds isolated from the six organs ranges from 25 to 64. There are 3 common compounds in the six organs, i.e. Dodecanoic acid, 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester and n-Hexadecanoic acid. This accounts for 12.0% of total hydrophilic volatile compounds for the leaf and 4.7% for the flower with an average of 8.4% for all the six organs, indicating the little commonality of the six organs. Each organ also has its exclusive compounds which are not found in any other organ. The percentage of exclusive compounds follows the order of flower > fruit > stem skin > root > stem > leaf. The flower has the largest number and percentage of the exclusive compounds, 40 and 62.5%, respectively. The leaf has the smallest number and percentage of the exclusive compounds, 9 and 36.0%, respectively. The stem and stem skin display medium numbers of exclusive compounds. The average percentage of the exclusive compounds in the six organs was 49.8%, nearly half, indicating strong organ specificity. These results provide evidence to support the practice of the traditional herbal medicine to treat the diseases using either the whole plant or part of the plants depending on which part contains the substances that can be used for therapeutic purposes.

Table 7: The number and percentage of the common and exclusive hydrophilic volatile compounds identified from the six organs.

Table 8 presents the number of overlapping compounds, overlapping percentage and overlapping index. The stem and stem skin share the largest number (15) of overlapping compounds. The overlapping percentage is calculated to be 32.6% for the stem and 35.7% for the stem skin. The smallest number (5) of overlapping compounds are found between root and fruit, leaf and fruit. The percentage of overlapping compounds between each of the two organs ranges from 10.9% to 44.0%, which is relatively small, further demonstrating substantial component differences among the different organs. The overlapping index is used to reveal the similarity among the organs. Two organs share the same number of overlapping compounds, but the overlapping index could be different if the total number of the hydrophilic volatile compounds differs. The more total number of the hydrophilic volatile compounds, the less the percentage of the overlapping compounds and smaller the overlapping index. That is why the average overlapping indices between the two organs is introduced to normalize the difference. In addition, total average overlapping indices is derived to calculate the mean of the average overlapping indices between each organ and the other five organs. Based on Table 8, the total average overlapping indices for each organ follows the order of stem > stem skin > root > leaf > flower > fruit. The total average overlapping indices for the stem is the greatest at 3.056, indicating the stem share the maximum similarity with the plant. The total average overlapping indices for the fruit was the smallest at 1.090, indicating that the fruit share the minimum similarity with the plant. And there is not much difference in the average overlapping indices between fruit and the other five organs. Except fruit, the average overlapping indices between each of the two organs correlate well to their physical proximity. The root, stem and stem skin are the organs that the plant survive and grow, and their total average overlapping indices are greater than 2.5. The overlapping index differences among these three organs are small, and they share the most in common. As an evergreen plant, the leaf is symbiotically related to the plant although the relationship between each leaf and the plant is cyclical, so the leaf is secondarily related to the plant. The flower and fruit are also cyclically related to the plant and have the most distant relationship. The leaf, flower and fruit are necessary but not survival organs for the growth of the plant. The relationship between the organs and the plant generated from the analysis of the hydrophilic volatile compounds is consistent with their biological function.

Table 8: The number of overlapping compounds, overlapping percentage and overlapping index.

Conclusion

The root, stem, stem skin, leaf, flower and fruit of the Ilex cornuta Lindl. & Paxton contain hydrophilic volatile compounds that are evenly distributed in the water phase of the various organs of the plant and can volatilize with water vapor. The number and type of hydrophilic volatile compounds vary from organ to organ. There is only a small number of common compounds among the six organs and the number of overlapping compounds between each of the two organs is also relatively small. In addition, there are large number of exclusive compounds from each organ. Therefore, it is possible to identify the plant through the assessment of the hydrophilic volatile compounds isolated from each individual organ.

In conclusion, we found that hydrophilic volatile metabolites are a class of natural products that are rarely investigated but constitute a significant part of the plant chemical composition. Chemical profiling of these secondary metabolites could provide a valuable tool for identification and authentication of the plant samples, as well as resolving taxonomic problems and understanding the chemically mediated biological phenomena.

References

1. Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, et al. (2007) Cryptic species as a window on diversity and conservation. Trends Ecol Evol 22: 148-155. [crossref]
2. Heinrichs J, Kreier HP, Feldberg K, Schmidt AR, Zhu RL, et al. (2011) Formalizing morphologically cryptic biological entities: New insights from DNA taxonomy, hybridization, and biogeography in the leafy liverwort Porella platyphylla (Jungermanniopsida, Porellales). Am J Bot 98: 1252-1262. [crossref]
3. Ludwiczuk A (2014) Fingerprinting of secondary metabolites of liverworts: chemosystematic approach. J of AOAC Int 97: 1234-1243.
4. Wink M (2003) Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry 64: 3-19. [crossref]
5. Liu K, Abdullah AA, Huang M, Nishioka T, Altaf-Ul-Amin M, et al. (2017) Novel Approach to Classify Plants Based on Metabolite-Content Similarity. BioMed Res Int. doi: 10.1155/2017/5296729
6. Ghaste M, Narduzzi L, Carlin S, Vrhovsek U, Shulaev V, et al. (2015) Chemical Composition of Volatile Aroma Metabolites and Their Glycosylated Precursors that Can Uniquely Differentiate Individual Grape Cultivars. Food Chem 188: 309-319. [crossref]
7. Peters K, Treutler H, Doll S, Kindt ASD, Hankemeier T, et al. (2019) Chemical Diversity and Classification of Secondary Metabolites in Nine Bryophyte Species. Metabolites 9: 222. [crossref]
8. Staszek D, Orlowska M, Rzepa J, Wrobel MS, Kowalska T (2014) Fingerprinting of the Volatile Fraction from Selected Thyme Species by Means of Headspace Gas Chromatography with Mass Spectrometric Detection. J of AOAC Int 97: 1250-1258. [crossref]
9. Tundis R, Peruzzi L, Menichini F (2014) Phytochemical and biological studies of Stachy Species in Relation to Chemotaxonomy: A Review. Phytochemistry 102: 7-39. [crossref]
10. Peñuelas J, Llusià J (1999) Seasonal emission of monoterpenes by the Mediterranean tree Quercus ilex in field conditions: Relations with photosynthetic rates, temperature and volatility. Physiol Plant 105: 641-647.
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15. Berlinck RGS, Monteiro AF, Bertonha AF, Bernardi DI, Gubiani JR, et al. (2019) Approaches for the isolation and identification of hydrophilic, light-sensitive, volatile and minor natural products. Nat Prod Rep 36: 981-1004. [crossref]

DOI: 10.31038/IMROJ.2021622

Abstract

This present study is aimed at determining phytochemical constituents with the aid of GCMS technique and in vitro screening of crude methanol extract of the leaves of Loranthus micranthus Linn parasitic on Citrus sinensis tree sourced from Nsukka, in the eastern part of Nigeria for their antimicrobial activity against six human pathogens. Preliminary phytochemical screening was carried out on the crude extract. The results of the phytochemical analysis showed positive for tannins, flavonoids, steroids, alkaloids, triterpenes/sterols, glycosides and saponins. GCMS analysis revealed nine bioactive compounds in the crude methanol extract of Loranthus micranthus, which includes; Benzoic acid, 3,4,5-trimethoxy-, trimethylsilyl ester, 1,3,5-Triazine, 2,4,6-tris[(trimethylsilyl)oxy]-, Gallic acid, Beta-Amyrin, Beta-Sitosterol, Lup-20(29)-en-3-one. The crude methanol extract of Loranthus micranthus was subsequently partitioned into four solvents to obtain n-hexane, chloroform, ethyl acetate and methanol fractions respectively. The crude extract and the four fractions were observed for antimicrobial activities on the following pathogens; Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Aspergillums niger, Staphylocoocus aureus and Candida albicans at various concentrations using agar diffusion method. The results of the antimicrobial activity proved the ability of the crude methanol extract and the obtained fractions of Loranthus micranthus to inhibit these pathogens. Different bacterial species and fungi exhibited different sensitivities with variable extent towards the extract/fractions. The order of activity against selected bacteria was Candida albicans> Bacillus subtilis> Staphylococcus aureus> Escherichia coli. Only the crude methanol extract at a concentration of 25 mg/ml inhibited Pseudomonas aeruginosa. While Aspergillums niger was resistant to both the extract and fractions. The n-hexane and the methanol fractions of Loranthus micranthus exerted maximum antimicrobial activity against Candida albicans with an MIC of 1.25 and 0.625 mg/ml respectively. An indication that the plant possesses very potent antifungal activities The MIC for Bacillus subtilis was 1.25 for n-hexane and methanol fractions respectively. There is good evidence that the plant is more active against gram positive bacteria.

Keywords

Antimicrobial, Gas chromatography-Mass spectrometry analysis, Loranthus micranthus Linn, Phytochemical

Introduction

Citrus sinensis belongs to the Rutaceae family, commonly known as sweet orange, which is a much sought after fruit worldwide with immense cultural and historical significance. The fruit’s origin dates back to its mention in the Chinese literature of 314 BC [1]. Orange trees thrive in both tropical and subtropical climates, making the plant; one of the most cultivated globally [2]. In the last decade, sweet orange accounted for 70% of the citrus fruits cultivated [2]. Currently, the production of oranges has hit 73 million tonnes, with Brazil, China and India leading in the production of oranges [3]. The fruit is a major source of vitamin C, which led to the massive cultivation of oranges along trade routes of European sailors in the discovery age. There has been numerous reports on the bioactivities of sweet oranges by many scholars. The anti-oxidant activities have received immense attention; similarly, the antimicrobial activities of the essential oil, the juice and the peels have equally been documented. However, the bioactivity of the mistletoes parasitic on oranges has very scanty reports, owing to the obvious fact that orange tree rarely bear host to parasitic plants such as mistletoe. A literature search revealed that the anti-microbial activities of Loranthus micranthus from Citrus sinensis has not been carried out, despite the plethoral of anti-microbial activities attributed to Citrus sinensis. While the mistletoes of many host trees such as Pentaclethra macrophylla, Persea americana, Kola nitida, Kola acuminata and Hevea brasiliensis have been investigated and shown to possess numerous activities, which includes: antioxidant, antimicrobial, antidiarrhoeal, immunomodulatory, antidiabetic, antihypertensive, and hypolipidemic activities [4]. The Citrus sinensis mistletoe has not been investigated.

In my previous work, it was observed that mistletoes parasitic on the host trees with significant bioactivity possessed some of the reported activities of their host trees. While the mistletoes parasitic on the host trees with few significant bioactivities also showed less significant bioactivities [5]. In this current work, the chemical characterization and antimicrobial evaluation of Loranthus micranthus (LM) parasitic on Citrus sinensis is reported.

Materials and Methods

Plant Material

Fresh leaves of LM parasitic on Citrus sinensis were obtained in Oba in Nsukka LGA of Enugu State. The plant was identified by a botanist in the Department of Botany, University of Nigeria, Nsukka, after which a voucher specimen was deposited in the department.

Extraction Procedure

The LM leaves were dried at room temperature under a shade, and pulverized. Weighed quantities of the LM were extracted with aqueous methanol using soxhlet extractor. The obtained methanol extract was subsequently partitioned into the following four solvents: N-hexane, Chloroform, Ethyl acetate and Methanol sequentially.

Preliminary Screening

The phytochemical constituents of the methanol extracts were determined according to the method prescribed by Trease and Evans [6].

GC-MS Analysis of the Crude Extracts

The gas chromatography mass spectrometry (GC-MS) analysis of the crude methanol extract of the leaves of Loranthus micranthus parasitic on C. sinensis was quantitatively determined using an Agilent 7890B GC system coupled with an Agilent 5977A MSD with a Zebron-5MS column (ZB-5MS 30 m × 0.25 mm × 0.025 μm) (5%-phenylmethylpolysiloxane). The GC-grade helium served as the carrier gas at a constant flow rate of 2 mL/min. The crude extract was dissolved with ethanol and filtered before use. The column temperature was maintained at 60°C and gradually increased at 10°C per minute until a final temperature of 300°C was reached. The time taken for the GC-MS analysis was 30 min. The compounds were identified based on computer matching of the mass spectra with the NIST 11 MS library (National Institute of Standards and Technology library).

Materials for Anti-microbial Assay

Equipment: Microscope, autoclave, incubator, wire loop, Bunsen burner, markers, petri dish test tubes and McCartney bottle.

Reagents

Nutrient agar and broth, Sabourand’s dextrose agar and broth, dimethyl sulfoxide (DMSO), sterile distilled water.

Test Organisms

All organisms used were clinical isolates obtained from the laboratory of pharmaceutical microbiology, University of Port Harcourt. The research utilized two gram positive bacteria, two gram negative bacteria and two fungi. The organisms were; Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Aspergillums niger, Staphylocoocus aureus and Candida albicans. The organisms were sub cultured and standardized before use.

Anti-Microbial Assay Method

Agar diffusion by cup plate method was adopted as described [7]. The entire agar used was prepared according to the manufacturer’s specification. The glass wares were thoroughly washed and sterilized, suspensions of the test organisms were made and 0.05 ml of 0.5 McFarland standard of the test organism concentration were dispensed each in the plate and 20 ml of the sterile molten agar each were added, mixed and allowed to set/gel, using two fold serial dilution. Different concentration of the methanol extract, the fractions and standard antibiotic used were made. Holes were bored on the agar using cock bore and labeled properly. 0.05 ml of each concentration was added into the respective holes with the aid of sterile syringe and allowed to diffuse for 15-20 minutes, then incubated at 37°C (bacterial), 25°C (fungi) for 24 hours. The zone of inhibition was determined against concentration with the aid of a meter rule to the nearest millimeter. Positive control for bacteria was Ciprofloxacin 100 mg/100 ml. Ketoconazole at a dose of 100 mg/ml served as the positive control for the fungi. The extract and fractions were solubilized in DMSO. A concentration of 50 mg/ml of the plant extracts were used for the antimicrobial assay. This concentration was serially diluted seven times to yield a minimal concentration of 0.78125 mg/ml. The MIC of the fractions which showed activity, were determined using the already diluted concentrations, 10 mg/ml, 5 mg/ml, 2.5 mg/ml, 1.25 mg/ml, 0.625 mg/ml 0.3125 mg/ml and 0.15625 mg/ml. The MIC was obtained as the least concentration that had a zone of inhibition.

Statistical Analysis

Each test was carried out in triplicate. The values were expressed as mean ± standard error of mean (SEM). The Dunnett one way analysis (ANOVA) was used to determine the significant differences among all columns against control and the P value < 0.05 was considered as significant. All statistical analysis was performed using Graph Pad Prism version 8.0 software.

Results

Table 1 shows the result of phytochemical analysis of the crude methanol extract of LM. The pharmacologically important classes of secondary metabolites are contained in the mistletoe. There is a heavy presence of flavonoids, saponins and tannins in the plant compared to the phytoconstituents. Similarly, the phytochemical analysis of the peels of C. sinensis have been documented to possess alkaloid, flavonoids, terpenoids, reducing sugars, saponins, tannins and amino acid [8].

Table 1: Results from the Phytochemical Screening.

The GCMS chemical characterization of the crude methanol extract was carried out and the results presented in Table 2. Nine compounds were identified from a spectral match with the NIST library of the equipment. There were some unidentified peaks in the GCMS chromatogram, but of the nine identified compounds, some pharmacological-active compounds with antimicrobial activities were recorded.

Table 2: GCMS Analysis Results.

The antimicrobial activities of the crude methanol extract are disclosed in Table 3. The results showed that the extract had pronounced anti-fungal activity. The results further demonstrated that the extract had less impact on gram negative bacteria in comparison to gram positive bacteria.

Table 3: Antimicrobial assay result of the crude Methanol extract.

The fractions obtained from the crude methanol extract were evaluated for their antimicrobial activities at a lower concentration to determine their potency. These results obtained are presented in Table 4. Significantly, all the fractions had no activity against P. aeruginosa and A. niger. However, various degrees of inhibitions were recorded against the tested pathogens.

Table 4: Inhibition Zone Diameter Exhibited By The Fractions At Different Concentrations.

Ciprofloxacin: Positive control for bacteria; Ketoconazole: Positive control for Fungi; DMSO: Negative control; +: No inhibition.

The analysis of the MIC of the crude methanol extract and fractions are portrayed in Table 5. The key observation is that both the crude extract and the fractions exercised antifungal activities against C albicans. Except the crude methanol extract at a concentration of 25 mg/ml, all the fractions had no activity over P. aeruginosa.

Table 5: MIC of Crude methanol Extract and Fractions.

Discussion

The citrus mistletoe is rich in flavonoids which are known to demonstrate antibacterial, anti-inflammatory and antifungal activities [9]. The presence of phenolic compounds has been attributed to antioxidant activities [10]. Flavonoids have both antifungal and antibacterial activity and anti-inflammatory properties [9]. Similarly, therapeutic benefits from alkaloids [11]and saponins [12] have been documented in literature.

The GCMS analysis of the Crude methanol extract revealed a number of peaks from which nine compounds were identified as Benzoic acid, 3,4,5-trimethoxy-, trimethylsilyl ester; 1,3,5-Triazine, 2,4,6-tris[(trimethylsilyl)oxy]-; Benzoic acid, 3,4,5-tris(trimethylsiloxy)-, trimethylsilyl ester (gallic acid); Beta-Amyrin trimethylsilyl ether (Beta-Amyrin); Beta-Sitosterol trimethylsilyl ether (Beta-Sitosterol); Lup-20(29)-en-3-one; Antra-9,10-quinone, 1-(3-hydrohy-3-phenyl-1-triazenyl)-; 4-Dehydroxy-N-(4,5-methylenedioxy-2-nitrobenzylidene)tyramine and 2-Thiophenecarboxylic acid, 4-methyl-5-nonadecyl-, methyl ester. It was discovered that the citrus mistletoe contained lupine-type triterpenes which have demonstrated significant cytotoxicities on human leukemias, melanomas and neuroblastomas. One analogue- Lup-28-al-20(29)-en-3-one, a close structural analogue of Lup-20(29)-en-3-one was the most bioactive [13].

Again the presence of gallic acid and its derivative Benzoic acid, 3,4,5-trimethoxy-, trimethylsilyl ester, strongly supports the antimicrobial activity of this mistletoe. Previous studies have documented the antimicrobial activities of gallic acid derivatives on Potato Bacterial Wilt Pathogen with 3,5-dihydroxy-4-methoxybenzoic acid shown to be the most potent one with a MIC value of 0.47 mg/ml [14].

Beta-amyrin (β-amyrin), another bioactive compound present in citrus mistletoe has been evaluated on clinical pathogens. The activity when compared with standard drugs was found to be comparable with the standard drug used [15].

β-sitosterol is the major phytosterols, found abundantly in plants is also present in citrus mistletoes. Many in vitro and in vivo studies have demonstrated various biological actions such as antimicrobial, anti – inflammatory, analgesic, immunomodulatory, anticancer, lipid lowering effect, anxiolytic & sedative effects, hepatoprotective, protective effect against NAFLD and respiratory diseases. Other effects include: wound healing effect, antioxidant and anti-diabetic activities [16]. The antimicrobial studies of novel series of 2,4-bis(hydrazino)-6-substituted-1,3,5-triazine and their Schiff base derivatives was carried using S. aureus, and E. coli among the pathogens evaluated. The 1,3,5-triazine were found to demonstrate significant activity on bacteria in microgram/ml range, but no antifungal activity was observed. It may be suggested that the presence of 1,3,5-triazine in the mistletoe contributed to the observed activity against the gram positive bacteria [17].

Conclusion

The results of the analyses shown earlier have demonstrated that the leaves of L. micranthus Linn parasitic on Citrus sinensis possess very potent antifungal activities against C. albicans and thus could be used as a therapeutic preparation in treatment of fungal Infections. Also, from the results the fractions displayed varying degree of antimicrobial activity against S. aureus, E. coli and B. subtilis. While P. aeruginosa and A. niger could not be inhibited by the fractions at the test concentrations. This suggests that the extracts had more activity against gram positive bacteria than against gram negative bacteria. The antimicrobial activity of the citrus mistletoe merits further research as the plant has potentials for development into antimicrobial agent that can be used in treating infections. Again, given that the plant contains some reported antimicrobial compounds, this might be exploited in the design and development fungicidal and bactericidal drugs.

Conflict of Interest

No potential conflict of interest was reported by the authors.

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